Thermal bioswitches and related genetic circuits, vectors, cells, compositions, methods and systems

ABSTRACT

Temperature sensitive transcriptional bioswitches and related genetic circuits and in particular bandpass and/or multiplex genetic circuits, vectors, cells, compositions methods and systems are described.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation under 35 U.S.C. § 120 of U.S.patent application Ser. No. 15/384,254, filed Dec. 19, 2016, which inturn claims priority to U.S. Provisional Application No. 62/269,715,entitled “Tunable Thermal Control of Transcription with EngineeredBioswitches” filed on Dec. 18, 2015, with docket number CIT-7388-P, eachof which is incorporated herein by reference in its entirety.

STATEMENT OF INTEREST

This invention was made with government support under Grant No.D14AP00050 awarded by the Department of the Interior. The government hascertain rights in the invention.

FIELD

The present disclosure relates to thermal bioswitches and relatedgenetic circuits, vectors and cells, as well as related compositions,methods and systems. In particular, the present disclosure relates tothermal bioswitches and related methods and systems to controlactivation and deactivation of genetic circuits in engineered cells.

BACKGROUND

Recent advances in synthetic biology are driving the development ofgenetically engineered cells for use in various applications whereincontrol of activation and deactivation one or more functions of theengineered cell is desired.

For example, a critical capability of cells engineered for use astherapeutic and diagnostic agents to treat human diseases, is theability to control activation of the therapeutic or diagnostic functionsof the engineered cells at anatomical and disease sites such as thegastrointestinal tract or tumors.

Despite development of molecular switches to control cells function,challenges remain for developing high-performance and/or tunablebioswitches to control gene expression in engineered cells in a widerange of applications including biomedical and industrial applications.

SUMMARY

Provided herein are thermal bioswitches and related genetic circuits,vectors and cells, as well as related compositions, methods and systemswhich can be used to provide a tunable control of one or more cellfunctions. In particular, in several embodiments thermal bioswitchesherein described can be used to provide thermally controllableultrasound, multiplexed and bandpass genetic circuit.

According to a first aspect, a coiled coil temperature sensitivetranscription factor is described. The coiled coil temperature sensitivetranscription factor comprises two monomer proteins configured to bindto one another to form a dimer in a target environment comprising atarget DNA polynucleotide having a DNA coding region under control of aDNA regulatory region, the dimer configured to have a DNA-bound stateand an DNA-unbound state with respect to specific binding of the dimerto the target DNA polynucleotide in the target environment

In the coiled coil temperature sensitive transcription factor, eachmonomer protein comprises a dimerization dependent DNA binding domainand a coiled coil temperature sensing domain, each having a N-terminusand a C-terminus, the N-terminus of the coiled coil temperature sensingdomain is covalently attached to the C-terminus of the DNA bindingdomain.In the dimerization dependent DNA binding domain of the coiled coiltemperature sensitive transcription factor, each monomer protein has anamino acid sequence configured to specifically bind the DNA regulatoryregion of the DNA polynucleotide in the target environment.In the coiled coil temperature sensing domain, the two monomer proteinsare configured to bind to one another in the target environment with abinding constant Kd≤100 nM in the DNA-bound state and ≥10 uM in theDNA-unbound state wherein

$\begin{matrix}{K_{d} = e^{(\frac{\Delta\; G}{RT})}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

in which R is the gas constant, T is the temperature of the targetenvironment and ΔG is the molar Gibbs free energy. The two monomerproteins of the coiled coil temperature sensing domain are furtherconfigured to bind to one another in the target environment with athermal Hill coefficient above 15 to form the dimer in a temperaturedependent manner.In the coiled coil temperature sensing domain, each monomer protein hasa coiled coil temperature sensing amino acid sequence having a lengthfrom 14 to 3200 amino acid residues, and a sequence

-   -   [X₁ X₂ X₃ X₄ X₅ X₆ X₇]_(a)    -   wherein X₁ is a hydrophobic amino acid, X₂ is a polar or charged        amino acid, X₃ is a polar or charged amino acid, X₄ is a        hydrophobic amino acid, X₅ is a polar or charged amino acid, X₆        is a polar or charged amino acid, and X₇ is a polar or charged        amino acid and n can be any integer between 2 to 457 (SEQ ID NO:        1 to SEQ ID NO: 456).        In the coiled coil temperature sensitive transcription factor,        the coiled coil temperature sensing amino acid sequence of each        monomer protein can comprise one or more insertions, deletions        or replacements with a percent variation with respect to SEQ ID        NO: 1 to SEQ ID NO: 456 from 0% to 20%. In the coiled coil        temperature sensing amino acid sequence of each monomer protein,        residues X₁ to X₇ are arranged in the temperature sensing amino        acid sequence to form consecutive uninterrupted series of 2 to        457 heptad repeats a, b, c, d, e, f, or g, at least two heptad        repeats of the 2 to 457 heptad repeats having a register in        which no amino acid is missing, and up to 455 heptad repeats a        register in which up to 5 consecutive amino acid residues are        optionally missing.        In the coiled coil temperature sensitive transcription factor,        the temperature sensing domain has a melting temperature Tm        defining a bioswitch temperature of the temperature sensitive        transcription factor (Tbs), the Tbs being a temperature of the        target environment at which the temperature sensitive        transcription factor is converted from the DNA bound state to        the DNA unbound state, with Tbs=Tm+0° C. to 5° C.        In some embodiments, each monomer protein of the coiled coil        temperature sensitive transcription factor is engineered to        replace at least one amino acid residue of the temperature        sensing domain to have a reduction or an increase in the melting        temperature Tm to provide a temperature sensitive transcription        factors with an increased or decreased Tbs.

According to a second aspect, a globular temperature sensitivetranscription factor is described. The globular temperature sensitivetranscription factor comprises two monomer proteins each having a lengthfrom 106 to 750 amino acids and is configured to bind to one another toform a dimer in a target environment comprising a target DNApolynucleotide having a DNA coding region under control of a DNAregulatory region, the dimer configured to have a DNA-bound state and aDNA-unbound state with respect to specific binding of the dimer to thetarget DNA polynucleotide in the target environment.

In the globular temperature sensitive transcription factor, each monomerprotein comprises a dimerization dependent DNA binding domain and aglobular temperature sensing domain, each having a N-terminus and aC-terminus, the N-terminus of the temperature sensing domain covalentlyattached to the C-terminus of the DNA binding domain.In the dimerization dependent DNA binding domain of the globulartemperature sensitive transcription factor, each monomer protein has anamino acid sequence configured to specifically bind the DNA regulatoryregion of the DNA polynucleotide in the target environment.In the globular temperature sensing domain, the two monomer proteins areconfigured to bind to one another in the target environment with abinding constant Kd≤100 nM in the DNA-bound state and ≥10 uM in theDNA-unbound state wherein

$\begin{matrix}{K_{d} = e^{(\frac{\Delta\; G}{RT})}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

in which R is the gas constant, T is the temperature of the targetenvironment and ΔG is the molar Gibbs free energy. The two monomerproteins of the coiled coil temperature sensing domain are furtherconfigured to bind to one another in the target environment with athermal Hill coefficient above 15 to form the dimer in the targetenvironment in a temperature dependent manner.

The globular temperature sensing domain has a length of 105 amino acids.and comprises an A bend, a B bend, a C bend, a D bend, an E bend, an Fbend, a G bend, an H bend, an I bend, a J bend, a K bend, an L bend, anM bend, a βA strand, a βB strand, a βC strand, a βD strand, a βE strand,a βF strand, an A turn, a B turn, a C turn, and a βA bridge linked oneto another by loop regions. The globular temperature sensing domain hasa sequence

(SEQ ID NO: 457) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀-X₅₁-X₅₂-X₅₃-X₅₄-X₅₅-X₅₆-X₅₇-X₅₈-X₅₉-X₆₀-X₆₁-X₆₂-X₆₃-X₆₄-X₆₅-X₆₆-X₆₇-X₆₈-X₆₉-X₇₀-X₇₁-X₇₂-X₇₃-X₇₄-X₇₅-X₇₆-X₇₇-X₇₈-X₇₉-X₈₀-X₈₁-X₈₂-X₈₃-X₈₄-X₈₅-X₈₆-X₈₇-X₈₈-X₈₉-X₉₀-X₉₁-X₉₂-X₉₃-X₉₄-X₉₅-X₉₆-X₉₇-X₉₈-X₉₉-X₁₀₀-X₁₀₁-X₁₀₂-X₁₀₃-X₁₀₄-X₁₀₅ whereinX₁ can be a polar residue defining an N-terminal residue;X₂ can be a polar residue forming the A bend;X₃ to X₅ can be polar or charged residues forming a loop;X₆ to X₈ can be polar amino acids forming the B bend;X₉ can be polar amino acid forming a loop;X₁₀ to X₁₃ can be any amino acids forming the βA strand;X₁₄ to X₁₅ can be any amino acids forming a loop;X₁₆ to X₁₉ can be polar residues forming the C bend;X₂₀ to X₂₂ can be polar or non-polar amino acids forming a loop;X₂₃ to X₂₄ can be polar amino acid residues forming the D bend;X₂₅ can be a non-polar amino acid forming a loop;X₂₆ to X₂₇ can be polar or ionic amino acids forming the E bend;X₂₈ to X₃₀ can be polar or non-polar amino acids forming a loop;X₃₁ to X₃₂ can be polar, non-polar or ionic amino acids forming the Fbend;X₃₃ can be any amino acid forming a loop;X₃₄ to X₃₇ can be non-polar amino acids forming the βB strand;X₃₈ to X₃₉ can be any amino acids forming a loop;X₄₀ to X₄₁ can be any amino acids forming the G bend;X₄₂ to X₄₄ can be any amino acids forming a loop;X₄₅ to X₄₆ can be any amino acids forming the A turn;X₄₇ can be a non-polar amino acid forming the H bend;X₄₈ to X₅₂ can be polar, non-polar, or ionic amino acids forming the βCstrand;X₅₃ can be any amino acid forming the I bend;X₅₄ to X₅₆ can be any amino acids forming the B turn;X₅₇ to X₆₀ can be any amino acids forming a loop;X₆₁ to X₆₄ can be any amino acids forming the βD strand;X₆₅ to X₆₆ can be any amino acids forming a loop;X₆₇ to X₆₉ can be any amino acids forming the J bend;X₇₀ can be any amino acids forming a loop;X₇₁ to X₇₃ can be any amino acids forming the βE strand;X₇₄ can be any amino acid forming a loop;X₇₅ to X₇₆ can be any amino acids forming the K bend;X₇₇ to X₇₈ can be polar amino acids forming the C turn;X₇₉ can be a polar amino acid forming the L bend;X₈₀ to X₈₁ can be polar amino acids forming a loop;X₈₂ can be a polar amino acid forming the βA bridge;X₈₃ to X₈₈ can be polar amino acids forming a loop;X₈₉ to X₉₆ can be polar or ionic amino acid residues forming the βFstrand;X₉₇ to X₁₀₀ can be polar or non-polar amino acids forming a loop;X₁₀₁ to X₁₀₃ can be polar amino acids forming the M bend;X₁₀₄ to X₁₀₅ can be polar or non-polar amino acids defining a C-terminalsegment,or a variant thereof in which any of the amino acid residues of SEQ IDNO: 457 is substituted with a ΔΔG of substitution greater than −0.5Rosetta Energy Unit (R.E.U) or lower than −0.5 Rosetta Energy Unit(R.E.U).In the globular temperature sensitive transcription factor, thetemperature sensing domain has a melting temperature Tm defining abioswitch temperature of the temperature sensitive transcription factor(Tbs), the Tbs being a temperature of the target environment at whichthe temperature sensitive transcription factor is converted from the DNAbound state to the DNA unbound state, with Tbs=Tm+0° C. to 5° C.In some embodiments, each monomer protein of the globular temperaturesensitive transcription factor is engineered to replace at least oneamino acid residue of the temperature sensing domain to have a reductionor an increase in the melting temperature Tm to provide temperaturesensitive transcription factors with an increased or decreased Tbs.

According to a third aspect, a method and variants obtainable therebyare described, the method directed to modify a bioswitch temperature Tbsof a coiled coil temperature sensitive transcription factor hereindescribed in a target environment, the coiled coil temperature sensitivetranscription factor comprising a coiled coil temperature sensing domainwith a melting temperature Tm with Tbs=Tm+0° C. to 5° C., the methodcomprising:

-   -   providing a coiled coil temperature sensitive transcription        factor herein described having a starting bioswitch temperature        Tbs₀ in the target environment and two monomer proteins        configured to form a temperature sensing domain in the target        environment with a starting melting temperature Tm₀; and    -   replacing in at least one monomer protein of the two monomer        proteins forming the temperature sensing domain    -   at least one of a hydrophobic amino acid in position a and a        hydrophobic amino acid in position d of at least one heptad        repeat in the temperature sensitive amino acid sequence of the        at least one monomer with residues configured to increase or        decrease hydrophobic packing between corresponding amino acid        residues in positions a and/or d of corresponding heptad repeats        in monomer proteins of the temperature sensing domain,    -   at least one of a polar or charged amino acid in position b, a        polar or charged amino acid in position e, and a polar or        charged amino acid in position g of at least one heptad repeat        in the temperature sensitive amino acid sequence of the at least        one monomer with a hydrophobic residue, and/or    -   at least one of a polar or charged amino acid in position e, and        a polar or charged amino acid in a position g of at least one        heptad repeat in the temperature sensitive amino acid sequence        of the at least one monomer with a residue configured to        increase or decrease coulombic repulsion between corresponding        amino acid residues in positions a, d, e and/or g of        corresponding heptad repeats in monomer proteins of the        temperature sensing domain,    -   the replacing performed to obtain a variant of the coiled coil        temperature sensitive transcription factor with a melting        temperature of the temperature sensing domain Tm_(m), lower or        higher than Tm₀ in the target environment, the obtained variant        having a bioswitch temperature Tbs_(m) lower or higher than Tbs₀        in the target environment.

According to a fourth aspect a method and variants obtainable thereby,the method directed to modify a bioswitch temperature Tbs of a globulartemperature sensitive transcription factor herein described in a targetenvironment, the globular temperature sensitive transcription factorcomprising a globular temperature sensing domain with a meltingtemperature Tm with Tbs=Tm+0° C. to 5° C., the method comprising:

providing a globular temperature sensitive transcription factor hereindescribed having a starting bioswitch temperature Tbs₀ in the targetenvironment and two monomer proteins configured to form a globulartemperature sensing domain in the target environment with a startingmelting temperature Tm₀; and

replacing in at least one monomer protein of the two monomer proteinsforming the temperature sensing domain,

at least one amino acid residue located in the globular temperaturesensing domain interface between the two monomer proteins selected fromX₁, X₂, X₂₀ to X₂₂, X₂₅, X₂₈ to X₃₀, X₃₁ to X₃₂, X₃₄ to X₃₇, X₄₇, X₄₈ toX₅₂, X₇₇ to X₇₈, X₇₉, X₈₀ to X₈₁, X₈₂, X₈₃ to X₈₈, X₈₉ to X₉₆, X₉₇ toX₁₀₀, and X₁₀₄ to X₁₀₅, and/or

at least one solvent exposed amino acid residue selected from X₃ to X₅,X₆ to X₈, X₉, X₁₆ to X₁₇, X₂₃ to X₂₄, X₄₂ to X₄₄, X₄₅ to X₄₆, X₅₃, X₅₄to X₅₆, X₅₇ to X₆₀, X₆₇ to X₆₉, and X₇₀,

the replacing performed to obtain a variant of the globular temperaturesensitive transcription factor with a melting temperature of thetemperature sensing domain Tm_(m) lower or higher than Tm₀ in the targetenvironment, the obtained variant having a bioswitch temperature Tbs_(m)lower or higher than Tbs₀ in the target environment.

According to a fifth aspect, an expression vector is described. Theexpression vector comprises a polynucleotide encoding for one or moretemperature sensitive transcription factors herein described, thepolynucleotide is comprised in the expression vector under control ofone or more regulatory sequence regions in a configuration allowing toexpress the one or more temperature sensitive transcription factorsencoded by the polynucleotide in presence of suitable cellulartranscription and translation factors. In some embodiments, the vectorcan further comprise a target DNA polynucleotide having a DNA codingregion under control of a DNA regulatory region, and the temperaturesensitive transcription factor is configured to have a DNA-bound stateand a DNA-unbound state with respect to specific binding of the dimer tothe target DNA polynucleotide in a target environment.

According to a sixth aspect, a temperature sensitive genetic circuit isdescribed to be operated in a target environment at at least two targettemperatures. The temperature sensitive genetic circuit comprises one ormore molecular components connected one to another by biochemicalreactions according to a circuit design. In the temperature sensitivegenetic circuit, at least one of the molecular components is atemperature sensitive genetic molecular component comprising a coiledcoil and/or globular temperature sensitive transcription factor hereindescribed having a bioswitch temperature Tbs in the target environmentequal to one of the at least two target temperatures. In the temperaturesensitive genetic circuit, each of the temperature sensitive geneticmolecular component is configured to activate or inhibit anothermolecular component of the genetic circuit at a temperature sensitivemolecular component bio switch temperature equal to the bioswitchtemperature Tbs of the coiled coil and/or globular temperature sensitivetranscription factor.

According to a seventh aspect, a temperature sensitive multiplexedgenetic circuit is described to be operated in a target environment atat least two different target temperatures. The temperature sensitivemultiplexed genetic circuit comprises one or more molecular componentsconnected one to another by biochemical reactions according to a circuitdesign. In the multiplexed genetic circuit at least two geneticmolecular components are temperature sensitive molecular components,each configured to activate or inhibit another molecular component ofthe genetic circuit at a temperature sensitive molecular componentbioswitch temperature.

In the multiplexed temperature sensitive genetic circuit, at least onefirst temperature sensitive molecular component is configured toactivate or inhibit a first another molecular component at a firstbioswitch temperature Tbs₁, and at least one second temperaturesensitive molecular component configured to activate or inhibit a secondanother molecular component at a second bioswitch temperature Tbs₂. Inthe multiplexed genetic circuit the first bioswitch temperature Tbs₁ isequal to one of the at least two different target temperatures of thetarget environment and the second bioswitch temperature Tbs₂ is equal toanother one of the at least two different target temperatures of thetarget environment. In the multiplexed genetic circuit, the firstanother molecular component is different from the second anothermolecular component and the first bioswitch temperature Tbs₁ isdifferent from the second bioswitch temperature Tbs₂. In someembodiments at least one of the temperature sensitive genetic molecularcomponents comprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs₁, or Tbs₂.

According to an eighth aspect, a temperature sensitive bandpass filteris described to be operated in a target environment at at least threetwo target temperatures forming a target temperature range, The bandpassfilter is configured to be operated within genetic circuit comprisingone or more molecular components connected one to another by biochemicalreactions according to a circuit design. The temperature sensitivebandpass filter comprises a first temperature sensitive geneticmolecular component configured to activate a first itself and/or a firstanother molecular component of the genetic circuit at a first bioswitchtemperature Tbs1 and to inhibit the first another genetic molecularcomponent at a second bioswitch temperature Tbs2. The temperaturesensitive bandpass filter further comprises a second temperaturesensitive genetic molecular components configured to inhibit the firsttemperature sensitive molecular component and/or the first anothermolecular component and to activate or inhibit a second anothermolecular component of the genetic circuit at the second bioswitchtemperature Tbs2.

In the temperature sensitive bandpass filter, the first bioswitchtemperature Tbs₁ is equal to one of the at least two different targettemperatures of the target environment, the second bioswitch temperatureTbs₂ is equal to another one of the at least two different targettemperatures of the target environment. In the bandpass genetic circuit,the first another molecular component is different from the secondanother molecular component and the first bioswitch temperature Tbs₁, isdifferent form the second bioswitch temperature Tbs₂. In someembodiments at least one of the temperature sensitive genetic molecularcomponents comprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs₁, or Tbs₂.

According to a ninth aspect, a temperature sensitive cell is describedto be operated in a target environment at at least two targettemperatures. The temperature sensitive cell comprises a temperaturesensitive genetic circuit herein described. In the temperature sensitivegenetic circuit, at least one of the genetic molecular components is atemperature sensitive genetic molecular components, each having abioswitch temperature Tbs_(C) in the cell equal to at least one of theat least two target temperatures. In some embodiments at least one ofthe temperature sensitive genetic molecular components comprises acoiled coil and/or globular temperature sensitive transcription factorherein described having a bioswitch temperature Tbs equal to Tbs_(C).

According to a tenth aspect, a temperature-sensitive therapeutic cell isdescribed. The temperature-responsive therapeutic cell comprises atemperature sensitive genetic circuit herein described comprising atleast one temperature sensitive molecular component and at least onetherapeutic molecular component. In the genetic circuit of thetherapeutic cell, the at least one temperature sensitive molecularcomponents is configured to activate or inhibit the at least onetherapeutic molecular component at a therapeutic bioswitch temperatureTbs_(T) In some embodiments bioswitch temperature Tbs_(T) is achieved inthe temperature sensitive therapeutic cell in response to a thermalstimulus. In some embodiments, the thermal stimulus is selected from ahost fever or external source of thermal energy such as focusedultrasound, infrared, magnetic particle hyperthermia. In someembodiments at least one of the temperature sensitive genetic molecularcomponents comprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs_(T).

According to a eleventh aspect, a temperature sensitive inactivable cellis described comprising a temperature sensitive genetic circuit hereindescribed in which at least one temperature sensitive molecularcomponent is configured to activate or inhibit at least one killermolecular component at an inactivating bioswitch temperature Tbs_(T) Insome embodiments the inactivating bioswitch temperature Tbs_(i) isachieved in the temperature sensitive therapeutic cell in response to adecrease in the cell temperature associated with a spatial translocationof the temperature sensitive inactivable cell. In some embodiments atleast one of the temperature sensitive genetic molecular componentscomprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs_(T).

According to a twelfth aspect, a composition is described. Thecomposition comprises one or more coiled coil and/or globulartemperature sensitive transcription factors, vectors, genetic circuitand/or temperature sensitive cells herein described together with asuitable vehicle.

According to a thirteen aspect, a method to control a biological processin an individual is described. The method comprises administering to theindividual one or more temperature sensitive cells herein describedcomprising a temperature sensitive genetic circuit herein described. Inthe method the temperature sensitive genetic circuit is configured toprovide an output interfering with the biological process in theindividual at a set target temperature between 34° C. and 44° C.

According to a fourteenth aspect, a method to treat a condition in anindividual, is described. The method comprises administering to theindividual one or more therapeutic temperature sensitive cells hereindescribed comprising a temperature sensitive genetic circuit hereindescribed. In the method the temperature sensitive genetic circuit isconfigured to provide a therapeutic output in the individual at a settarget temperature between 34° C. and 44° C.

According to a fifteenth aspect, a method to control cell viability in atemperature sensitive manner is described. The method comprisesproviding a temperature sensitive cell comprising one or moretemperature sensitive genetic circuits herein described comprising atleast one temperature sensitive molecular component configured toactivate at least one killer molecular component at an inactivatingbioswitch temperature Tbs₁, The method also comprises applying to thetemperature sensitive cell the inactivating bioswitch temperature Tbs₁for time and under condition to allow activation of the at least onekiller components by the at least one temperature sensitive molecularcomponent and to result in death of the temperature sensitive cell.

In some embodiments of the temperature sensitive transcription factors,genetic circuits, vectors, cells, and related methods and systems atarget temperature of the environment Tbs can be selected from 34 to41<33° C. (including ambient environment), 33-34° C. (including skintemperature) 34-36° C. (including hypothermic core temperature), 36-38°C. (including human physiological temperature), 38-40° C. (includingmild fever in humans, 40° C.-42° C. (including severe fever in humans),39° C.-45° C. (including applied hyperthermia in humans (e.g. HIFU))

Temperature sensitive transcription factors, genetic circuit, cells andrelated vectors compositions, methods and systems herein described,allow in several embodiments to spatially and/or temporally controlactivation of cellular functions in a cell through a thermally regulatedcoupling of endogenous or applied signals to cellular function.

Temperature sensitive transcription factors, genetic circuit, cells andrelated vectors compositions, methods and systems herein described,allow in several embodiments to spatially and/or temporally controlactivation of cellular functions of a systemically administeredmicrobial therapy to a specific anatomical site, such as a deep-seatedtumor or section of the gastrointestinal tract that would be difficultto reach with other triggers (e.g. optogenetic).

Temperature sensitive transcription factors, genetic circuit, cells andrelated vectors compositions, methods and systems herein described,allow in several embodiments to locally target therapeutic effects ofcell therapy provided via systemic administration, minimizing therelated side effects.

Temperature sensitive transcription factors, genetic circuit, cells andrelated vectors compositions, methods and systems herein described,allow in several embodiments to combine thermally-triggered geneexpression in vivo with genetically encoded genomic or proteomic toolsto enable the study of cellular signaling within the context ofmammalian hosts.

Temperature sensitive transcription factors, genetic circuit, cells andrelated vectors compositions, methods and systems herein described,allow in several embodiments to program controlled responses tomammalian host temperature, (e.g. production of a therapeutic agent by atemperature sensitive cell in response to a host fever or runawayinflammation, or expression a therapeutic gene by a temperaturesensitive cell only within a specified temperature range).

Temperature sensitive transcription factors, genetic circuit, cells andrelated vectors compositions, methods and systems herein described,allow in several embodiments to restrict bacterial viability within athermal range (e.g. to confine the activity of genetically engineeredmicrobes to the in vivo environment of a mammalian host and therebylimit the potential for environmental contamination, or to obtain agreater efficiency of multilayered and multi-input containment circuitsin preventing mutational escape).

Temperature sensitive transcription factors, genetic circuit, cells andrelated vectors compositions, methods and systems herein described, canbe used in connection with various applications wherein controlledoutput of a genetic circuit is desired. For example, temperaturesensitive transcription factors, genetic circuit, cells and relatedvectors compositions, methods and systems herein described can be usedto provide spatially and/or temporally controlled expression oftherapeutics in medical applications, drug research and manufacturing,biological synthesis of chemicals and proteins such as enzymes orcatalysts or polymers, as well diagnostics and/or clinical applications.Additional exemplary applications include uses of temperature sensitivetranscription factors, genetic circuit, cells and related vectorscompositions, methods and systems in several fields including basicbiology research, applied biology, bio-engineering, bio-energy, medicalresearch, medical diagnostics, therapeutics, bio-fuels, and inadditional fields identifiable by a skilled person upon reading of thepresent disclosure.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description and theexamples, serve to explain the principles and implementations of thedisclosure.

FIGS. 1A-1H show examples of optical density (OD) measurements ofcultures of E. coli transformed with the indicated plasmid constructs.OD was measured at at 600 nm, at the indicated temperatures,corresponding to the OD₆₀₀ measurements for thermal induction profilesreported in Examples. Absorbance units (A.U.) of OD shown areblank-subtracted (bacterial culture minus culture media only)measurements of OD₆₀₀ in 90 μL volumes in clear-bottom 96 well plates,corresponding to an optical path length of approximately 1.4 mm. OD₆₀₀measurements in FIG. 1A correspond to FIG. 2B OD₆₀₀ measurements in FIG.1B correspond to FIG. 2C; OD₆₀₀ measurements in FIG. 1C correspond toFIG. 2E; OD₆₀₀ measurements in FIG. 1D correspond to FIG. 2F; OD₆₀₀measurements in FIG. 1E correspond to FIG. 6D; OD₆₀₀ measurements inFIG. 1F correspond to FIG. 6B; OD₆₀₀ measurements in FIG. 1G correspondto FIG. 8C; OD₆₀₀ measurements in FIG. 1H correspond to FIG. 8G. ‘BG’indicates measurement of E. coli transformed with a non-fluorescentconstruct.

FIGS. 2A-2F show exemplary results of characterization ofhigh-performance thermal bioswitches. FIG. 2A shows diagrams of examplesof constructs used to assay the performance of temperature sensitiverepressors (TSR, top) and heat shock promoters (HSP, bottom), wherelarge arrows indicate expressed genes (green fluorescent protein, GFP;TSR) and small arrows indicate promoters (pTSR, pLacI, pHSP). ‘T7 term’and a ‘T’ symbol indicate a T7 RNA polymerase transcriptionalterminator. The ‘flat-headed line’ symbol above the pTSR promoterindicates transcriptional repression by TSR. The specific repressors andpromoters assayed are listed to the right of the diagrams of expressionconstructs. The illustration of bacteria before and after a temperaturechange (ΔT) depicts de-repression of a thermally-gated promoter andexpression of GFP. FIG. 2B shows a graph of examples of OD-normalizedfluorescence (normalized fluorescence units, NFU) after 12 hours ofthermal induction for the constructs shown in FIG. 2A. Minimumfluorescence (F_(min)) represents expression at 31.4° C. F_(max) is themaximum fluorescence intensity measured for each construct, measured upto 45.7° C. The fold change between F_(min) and F_(max) is shown aboveeach sample. Where not seen, error bars are smaller than the symbol. N=4for TSRs and N=3 for HSPs. *The TcI F_(min) is reported from measurementat 34.2° C. because expression at lower temperatures was below thedetection limit of the assay. FIG. 2C shows a graph of exemplaryOD-normalized fluorescence from the TlpA- and TcI-regulated constructsas a function of induction temperature for a fixed duration of 12 hours.FIG. 2D shows a graph of exemplary OD-normalized fluorescence as afunction of thermal induction duration at the maximal inductiontemperature for the TlpA and TcI constructs. FIG. 2E and FIG. 2F showimages of examples of OD-normalized fluorescence landscapes for TlpA andTcI-gated constructs, respectively, as a function of both incubationtemperature and duration, where data shown are interpolated from an 8×8sampling matrix of these variables. All samples in FIGS. 2D-F weremaintained at 30° C. after the indicated period of thermal induction fora total experimental duration of 24 hours prior to measurement.

FIGS. 3A-3B show graphs of examples of prevalence of repressor sequencesin bacteria. FIG. 3A shows a graph of National Center for BiotechnologyInformation (NCBI) Basic Local Alignment Search Tool (BLAST) searchresults for the wild type tlpA, cI, tetR, and lacI genes showing thecumulative number of hits obtained. The NCBI nucleotide collection wassearched with the source organism restricted to bacteria. Cloningvectors, synthetic constructs, and individual gene sequences wereomitted; genomic and naturally occurring plasmid sequences wereretained. Sequences with alignment lengths of less than 90% of the wildtype protein sequence were not included. The lacI gene is distributedthroughout many commonly utilized E. coli strains such as Nissle 1917and BL21, whereas the cI gene is found in less widely used E. colistrains. FIG. 3B shows a graph of examples of the number of bacterialspecies in which the selected repressors are found. Data were obtainedas in FIG. 3A and substrains were binned together. TlpA is largelyrestricted to S. enterica and cI to E. coli; tetR and lacI can be foundin a larger number of bacterial species.

FIGS. 4A-4E show exemplary results indicating mechanisms andbidirectional activity of the TlpA operator. FIG. 4A shows a graph ofexemplary OD-normalized expression of the GFP reporter gene under thecontrol of TlpA, LacI^(q), and TlpA_(SP) (in which nucleotides withinthe Pribnow box of the operator are shuffled) at the indicatedtemperatures. FIG. 4B shows an image of a gel showing exemplary resultsof an electromobility shift assay using a FAM-labeled pTlpA operatoroligonucleotide visualized with blue epifluorescent illumination. Thebands in lanes 3 and 5 indicated by white arrowheads represent pTlpAoligonucleotides bound to the E. coli σ ⁷⁰-RNAp holoenzyme and TlpA,respectively, demonstrating association of the operator with both the E.coli σ ⁷⁰-RNAp holoenzyme and TlpA. In contrast, scrambled TlpA operator(shown in lanes 4 and 6) fails to associate with these proteins. TheTlpA and σ⁷⁰-RNAp concentrations used in this experiment (1 μM and 0.18μM, respectively) were similar to previous literature [1, 2]. FIG. 4Cshows a graph of exemplary GFP expression driven by the TlpA operator inthe canonical (Forward) and flipped (Reverse) orientations at 44.1° C.FIG. 4D shows a graph of exemplary thermal induction profiles for GFPexpression under the control of forward and reverse-oriented TlpAoperator, where each curve is self-normalized to its maximalfluorescence intensity. FIG. 4E shows a diagram of the proposedmechanism of TlpA-based thermal transcriptional regulation. Theillustration on the left side of the diagram shows the association of adimer of TlpA repressor with the TlpA operator, at temperatures belowthe de-repression temperature, preventing the association of theσ⁷⁰-RNAp holoenzyme with the TlpA operator containing a Pribnow box. Anincrease in temperature (ΔT) is proposed to cause an unwinding of thecoiled coil domain of TlpA (shown on the right side of the diagram) anddissociation of TlpA from the TlpA operator, permitting association ofthe σ⁷⁰-RNAp holoenzyme with the TlpA operator, regulating strongtranscription from the forward orientation of the promoter and weaktranscription in the reverse orientation.

FIG. 5A shows an illustration of an exemplary screening strategy used toidentify temperature-shifted repressor variants. A library of bacterialcolonies transformed with plasmids comprising mutated repressor proteinvariants is replica-plated and incubated at low temperature or hightemperature and fluorescence of colonies is visualized. The replicaplated colony indicated by the horizontal arrow shows no fluorescentreporter expression at low temperature, and shows reporter fluorescenceat high temperature, indicating de-repression at the high temperature.FIG. 5B shows a graph of exemplary self-normalized fluorescence/ODprofiles for a set of TlpA (solid lines) and TcI (dashed lines)bioswitches, demonstrating a range of transition temperatures. Here,‘self-normalized’ indicates that the maximal fluorescence/OD measurementfor each variant was given a value of 1.0.

FIGS. 6A-6D show exemplary results of tuning the transition temperatureof thermal bioswitches. FIG. 6A shows examples of fluorescent images ofreplica plates used to screen for TlpA variants turning on between 37°C. and 40° C. One colony selected for further analysis is indicated byan arrow and a pair of circles around the colony replica plated andincubated at the two temperatures. FIG. 6B shows a scatter plot ofexamples of TlpA variants plotted by their measured midpoint transitiontemperatures (T_(m)) and 10%-90% transition range (T_(10_90)), estimatedby linear interpolation. The shade of grey of each data point maps tothe change in fluorescence over the T₁₀₋₉₀ span. FIG. 6C shows a graphof exemplary OD-normalized fluorescence of novel TlpA variants.OD-normalized fluorescence values of TlpA variants are also normalizedto wild-type, with the maximal wild-type value given a value of 1.0.FIG. 6D shows a graph of exemplary OD-normalized fluorescence of novelTcI variants normalized to wild-type. Scale bars 1 cm.

FIGS. 7A-7C show positions of mutations in selected exemplary variantsof TlpA. FIG. 7A shows a schematic of exemplary mutation positionswithin the predicted domain structure of TlpA₃₆ (P60L, D135V, K187R,K202I, L208Q) and TlpA₃₉ (D135V, A217V, L236F) The DNA binding domain isdepicted in dark grey and coiled-coil domain in light grey, asdelineated by Koski et al [3]. The figure is drawn to the scale of theprimary sequence. FIG. 7B shows ‘helical wheel’ diagrams of positions ofmutations in TlpA₃₆ within the predicted structure of the coiled-coilinterface as viewed down the long axis of the helix, with two TlpAproteins shown side-by-side, in a predicted dimer configuration of thetranscriptionally repressive state. For each helical wheel diagram, aportion of a predicted alpha-helical heptad repeat (labeleda-b-c-d-e-f-g) is shown, connected by progressively thinner straightlines shown in an N-terminal to C-terminal direction. A dashed line isshown between the ‘g’ of a heptad and the ‘a’ of a next heptad in aportion of the heptad repeat. Each helical wheel diagram shown depictsthree consecutive heptads, with three single-letter amino acid symbolsshown circled at each position in a heptad. The sequence of amino acidsin an N-terminal to C-terminal direction are shown at each position of aheptad, with the first heptad in the portion of the heptad repeat shownon the line of each large circle representing each alpha-helix; theamino acids of the second consecutive heptad are shown further out fromthe large circle, and the third consecutive heptad are shown outermostfrom each large circle. The lines comprised of small ‘x’ symbols in themiddle diagram of FIG. 7(b) indicate predictive inter-monomericrepulsive ionic interaction between the indicated R and K residues; allinter-monomeric dashed lines represent predicted energetically favorableionic interactions. The coil register was assigned based on consensusbetween previous literature [3] and the structure prediction serversCOILS [4], Paircoil2 [5], and LOGICOIL [6]. The images were producedusing DrawCoil 1.0 [7]. The P60L mutation is not shown because it fallsoutside of the predicted coiled-coil region. FIG. 7C shows ‘helicalwheel’ diagrams of positions of mutations in TlpA₃₉. Heptad repeatregister prediction and illustration were performed as in FIG. 7B. FIG.7D shows an example of a proposed TlpA variant mutated by swapping pairsof coulombically-attracting amino acid residues with those expected toexperience coulombic repulsion (indicated by inter-monomeric dashedlines between R and E residues) which is expected to shift thede-dimerization temperature threshold down. Amino acid mutations shownin FIG. 7(d) are E282, R283, R292, and E297. FIG. 7E shows an example ofa proposed TlpA variant mutated by swapping pairs ofcoulombically-repelling amino acid residues with those no longerexpected to experience coulombic repulsion (inter-monomeric linescomprised of small ‘x’ symbols between the indicated R and K residues)which is expected to shift the de-dimerization temperature threshold up.Amino acid mutations shown in FIG. 7(e) are R182 and K187. FIG. 7F showsan example of appending short charged sequences to TlpA protein so as toeffect coulombic repulsion. Here the short charged protein sequencesindicated by “DED” coulombically repel each other (indicated by an ‘x’symbol), which is expected to shift the de-dimerization temperaturethreshold down. FIG. 7G-7H shows positions of mutations in selectedexemplary variants of TcI. The crystal structure of the wild type lambdarepressor (Protein Data Bank (PDB) code 3BDN) was used as the homologymodel [8]. The original temperature-sensitizing mutation in cI⁸⁵⁷ A67Tis not shown. The M1V mutation is not depicted because residue 1 was notreported in the crystal structure. FIG. 7G shows a crystal structuremodel image depicting mutation positions (spheres) for the lambdarepressor variant TcI₃₈. FIG. 7H shows a crystal structure model imagedepicting mutation positions (spheres) within the TcI₄₂ variant. FIG. 7Ishows a zoomed-in view of part of the TcI crystal structural modelshowing an example of replacing polar residues at the TcI dimerizationcore interface with different (e.g. bulkier) polar residues that aresterically inhibited from forming polar contacts as efficiently as inthe original protein. The image shows a zoomed-in view of the K68Rmutation in the TcI₃₈ variant. The left panel shows the structure of TcIwith the original K68 residue of TcI. The right panel shows acomputational prediction of TcI structure with a K68R mutation based onthe Mutagenesis functionality of PyMOL software. FIG. 7J shows azoomed-in view of part of the TcI crystal structural model showing anexample of minimizing entropy of the folded structure by replacinggeometrically constrained residues with smaller residues containing lessdegrees of freedom while maintaining the energetic contribution of polarcontacts. The image shows a zoomed-in view of the K6N mutation in theTcI₄₂ variant. The left panel shows the structure of TcI with theoriginal K6 residue of TcI. The right panel shows a computationalprediction of TcI structure with a K6N mutation based on the Mutagenesisfunctionality of PyMOL software.

FIG. 8A shows a diagram illustrating an example of multiplexed thermalactivation. The graph shows simulated data of expression activity of GFP(grey) and RFP (black) under control of pTlpA and pR/pL promoters,respectively, as shown in the thermal logic circuits illustrated in FIG.8B, as a function of temperature. The illustrations of bacteria beforeand after temperature increases (arrows) show de-repression ofthermally-gated promoters, corresponding to the gene expression activityin the graphed simulated data below each bacterium, where, astemperature increases, GFP expression alone is activated first, followedby additional activation of RFP expression. FIG. 8B shows an exemplarythermal logic circuit diagram of the pCali2 plasmid, which contains GFPthermally gated by TlpA₃₆ and RFP thermally gated by TcI, where largearrows indicate expressed genes (GFP, tlpA₃₆, RFP and tcI) and smallarrows indicate promoters (pTlpA, pLacI, and pR/pL). ‘T7 term’ and a ‘T’symbol indicate a T7 RNA polymerase transcriptional terminator. The‘flat-headed line’ symbol above the pTlpA and pR/pL promoters indicatestranscriptional repression by TlpA₃₆ and TcI, respectively. FIG. 8Cshows a graph of exemplary expression (OD-normalized fluorescence units,NFU) of GFP (grey) and RFP (black) from pCali2-containing E. coli overthe indicated range of temperatures (following 12 hours of incubation ateach temperature). FIG. 8D shows exemplary images of transformed E. colibacterial culture plates with overlaid GFP and RFP fluorescence from E.coli transformed with the pCali2 plasmid (plated in a depiction ofgrass, circled and indicated with ‘i’ at 37° C. and ‘iii’ at 42° C.) andE. coli transformed with plasmids expressing only the components of thepCali2 plasmid that regulate GFP expression (plated in a depiction of atree, circled and indicated with ‘ii’ at 37° C. and ‘iv’ at 42° C.) orRFP expression (plated in a depiction of the Sun, circled and indicatedwith ‘v’ at 42° C.). At 42° C. the grass shows both GFP and RFPfluorescence (iii), indicating expression of both GFP and RFP at thistemperature. FIG. 8E shows a diagram illustrating an example of theactivity of a thermal band-pass filter. The graph shows simulated dataof expression activity of GFP (grey) and RFP (black) under control ofpTlpA and pR/pL promoters, respectively, as shown in the thermal logiccircuits illustrated in FIG. 8F, as a function of temperature. Theillustrations of bacteria before and after temperature increases(arrows) show de-repression of thermally-gated promoters, correspondingto the gene expression activity in the graphed simulated data below eachbacterium, where, as temperature increases, RFP expression alone isactivated first, followed by simultaneous activation of GFP expressionand repression of RFP expression. FIG. 8F shows an exemplary thermallogic circuit diagram of the pThermeleon plasmid, in which RFP isthermally gated by TcI₃₈ and also by the wild type cI repressor. GFP isgated by TlpA_(wt) on the same plasmid, which also weakly drives theexpression of cI_(wt) through a T7 terminator and weak ribosome bindingsite. Large arrows indicate expressed genes (GFP, tlpA_(Wt), cI_(wt),RFP and tcI₃₈) and small arrows indicate promoters (pTlpA, pLacI, andpR/pL). ‘T7 term’ and a ‘T’ symbol indicate a T7 RNA polymerasetranscriptional terminator. ‘Synthetic term’ and a ‘T’ symbol indicate asynthetic RNA polymerase transcriptional terminator (BBa_B 1002). The‘flat-headed line’ symbol above the pTlpA promoter indicatestranscriptional repression by TlpA_(wt) and the ‘flat-headed line’symbols above the pR/pL promoters indicates transcriptional repressionby both TcI₃₈ and cI_(wt). FIG. 8G shows a graph of exemplary expression(OD-normalized fluorescence units, NFU) of RFP and GFP frompThermeleon-containing E. coli over the indicated range of temperatures(following 12 hours of incubation at each temperature). FIG. 8H showsoverlaid GFP and RFP fluorescence images of plated E. coli containingthe pThermeleon plasmid cultured at 40° C. and 45° C. At 40° C., RFPfluorescence is dominant, while at 45° C., GFP fluorescence is dominant.Scale bars 1 cm.

FIGS. 9A-9E show examples of remote control of thermal bioswitches inbacterial agents using focused ultrasound. FIG. 9A shows an illustrationof an example of an in vitro focused ultrasound experiment: focusedultrasound is used to heat a target area of a bacterial culture lawn ofbacteria transformed with a plasmid encoding a thermal bioswitchcomprising GFP under control of TplA₃₆ and RFP regulated by TcI, througha tofu phantom (depicted as translucent) under MRI guidance, followed byfluorescent imaging. Patterned gene expression results from a thermalgradient (with the highest temperature in the middle of the ultrasoundfield) acting on the bioswitches. FIG. 9B shows an image of an MRI-basedtemperature map of the transformed E. coli culture lawn depicted in FIG.9A during steady-state ultrasound application, overlaid on a raw MRIimage of the phantom. FIG. 9C shows an example of a fluorescent image ofthe region targeted by ultrasound depicted in FIG. 9B, showingactivation consistent with a bacterial construct expressing GFP underthe control of TlpA₃₆ and RFP regulated by TcI, where the light greyspot in the middle corresponds to RFP expression at the focal point ofultrasound application (highest temperature in the thermal gradient) andthe dark grey circle at the penumbra of the focused ultrasound (wheretemperature is lower) corresponds to GFP expression. FIG. 9D shows anillustration of an example of an in vivo experiment, in which focusedultrasound is used to activate subcutaneously-injected bacteriatransformed with a plasmid encoding a thermal bioswitch regulating GFPexpression, at a specific anatomical site, for example subcutaneouslyinto hindlimbs (shown by circles with dashed lines), where ultrasoundapplication results in GFP expression (dashed circle filled in withgrey). FIG. 9E shows an exemplary image of a thresholded fluorescencemap of a mouse injected subcutaneously in both left and right hindlimbswith E. coli expressing GFP under the control of TlpA₃₆, followingultrasound activation at only the right hindlimb. Focused ultrasound atthe site indicated with a black arrowhead results in highestfluorescence level in the center of the focused ultrasound application(dark grey spot in center), surrounded by a light grey circle at thepenumbra of focused ultrasound application. Scale bars 2 mm (FIG. 9B andFIG. 9C) and 1 cm (FIG. 9E).

FIGS. 10A-10F show examples of programmed thermal bioswitch responses tomammalian host temperature. FIG. 10A shows an illustration of an exampleof an experiment showing fever-induced gene expression activation usinga thermal bioswitch and an example of a thermal logic circuit diagram ofa corresponding E. coli construct. In the illustration, a mouse isadministered with a bacterium transformed with a plasmid comprising GFPregulated by TplA₃₆, which represses GFP expression at sub-fevertemperature. After induction of fever (arrow) GFP expression isde-repressed, shown by a grey shaded area within the dotted circle inthe mouse on the right. In the thermal logic circuit diagram, largearrows indicate expressed genes (GFP, tlpA₃₆) and small arrows indicatepromoters (pTlpA, pLacI). ‘T7 term’ and a ‘T’ symbol indicate a T7 RNApolymerase transcriptional terminator. The ‘flat-headed line’ symbolabove the pTlpA promoter indicates transcriptional repression by TlpA₃₆.FIG. 10B shows an exemplary image of a thresholded fluorescence map of amouse that underwent fever induction after being injected subcutaneouslywith plasmids expressing TlpA₃₆- and TlpA_(wt)-regulated GFP into theleft and right hind limbs, respectively, showing a spot of fluorescenceon the left hindlimb. The highest fluorescence level (black area incenter of the spot) is surrounded by a light grey circle at thepenumbra, corresponding to a gradient of injected bacterialconcentration (highest at the center). FIG. 10C shows an exemplary imageof a thresholded fluorescence map of a mouse that was preparedidentically to the animal in FIG. 10B, but maintained at roomtemperature, and does not show a fluorescent spot in the left hindlimb.FIG. 10D shows an illustration of an example of a temperature-based hostconfinement strategy, and an exemplary circuit diagram of a thermal killswitch permitting bacterial survival only at temperatures above 36° C.In the illustration, a mouse is administered into the gastrointestinaltract with a bacterium transformed with a plasmid comprising a constructin which antitoxin CcdA fused to a degradation tag ssrA (encoded byccdA-ssrA) expression is de-repressed by TlpA₃₆. Within the mouse gut,at 37° C., antitoxin expression is de-repressed and the bacterium isalive. Following defecation (arrow; fecal pellets represented by darkovals), decreased temperature outside of the mouse (25° C.) results inrepression of antitoxin expression and death of the bacteria resultingfrom the expressed toxin CcdB (encoded by ccdB). In the thermal logiccircuit diagram, large arrows indicate expressed genes (CmR, ccdB,tlpA₃₆, and ccdA-ssrA) and small arrows indicate promoters (constitutivepromoter lac UV5; pTlpA). ‘T7 term’ and a ‘T’ symbol indicate a T7 RNApolymerase transcriptional terminator. The ‘flat-headed line’ symbolabove the pTlpA promoter indicates transcriptional repression by TlpA₃₆.CmR is used to reduce the expression of the promoter and the weak RBS isused to reduce leakiness of CcdA expression and reduce toxin CcdBexpression to prevent mutations FIG. 10E shows an example of a graph ofcolony counts from liquid cultures (colony forming units, CFU, per mL ofculture) of killswitch-containing cells and controls (containing notoxin system) after 24 hours of incubation at the indicated temperature.P-value=0.0002 for kill switch vs. control cells at 25° C. and p<0.0001for kill switch at 25° C. versus 37° C. FIG. 10F shows an example of agraph of colony counts in fecal samples freshly collected from N=5 mice5 hours after oral gavage of killswitch-containing E. coli or controls.The feces were incubated at a temperature representative ofpost-defecation conditions (25° C.), or incubated at 37° C. (Rescue).P-value=0.0067 for kill switch vs. control cells at 25° C. and p=0.0275for kill switch at 25° C. versus 37° C. Scale bars 1 cm.

FIGS. 11A-11B show the DNA and protein sequence of TlpA. FIG. 11A showsthe DNA sequence (SEQ ID NO: 460). FIG. 11B shows the single-letteramino acid code sequence of TlpA protein (SEQ ID NO: 461). Boldunderlined amino acid residues are mutated in the TlpA₃₆ variant, italicunderlined residues are mutated in the TlpA₃₉ variant, and the lowercaseunderlined amino acid residue is mutated in both the TlpA₃₆ variant andthe TlpA₃₉ variant. The ‘*’ symbol represents the stop codon at theC-terminus of the protein

FIGS. 12A-12B show the DNA and protein sequence of the TlpA₃₆ variant.FIG. 12A shows the DNA sequence (SEQ ID NO: 462). FIG. 12B shows thesingle-letter amino acid code sequence of TlpA₃₆ protein (SEQ ID NO:463). The ‘*’ symbol represents stop codon at the C-terminus of theprotein

FIGS. 13A-13B show the DNA and protein sequence of the TlpA₃₉ variant.FIG. 13A shows the DNA sequence (SEQ ID NO: 464). FIG. 13B shows thesingle-letter amino acid code sequence of TlpA₃₉ protein (SEQ ID NO:465). The ‘*’ symbol represents stop codon at the C-terminus of theprotein

FIG. 14 shows the amino acid sequence of TlpA (SEQ ID NO: 461) dividedinto heptad repeat portions ‘A’ through ‘M’ in the predicted coiled-coildomain, with the coil register of heptads assigned based on consensusbetween previous literature [3] and the structure prediction serversPaircoil2 [5] and LOGICOIL [6]. The register position within the heptadrepeat for each predicted heptad repeat portion ‘A’ through ‘M’ isindicated; for example “A starts at position b”. The predicted DNAbinding domain is also indicated (residues 1 to 69). The amino acidposition number within each heptad portion of the coiled coil domain andwithin the DNA binding domain is indicated above each upper row (grey),while the amino acid position number within the whole TlpA proteinsequence is indicated above each lower row (black). The symbol “*”indicates the stop codon at the C-terminus of the TlpA protein. Anarrowhead symbol ‘>’ at the end of each line indicate the direction ofthe amino acid sequence.

FIG. 15 shows the amino acid sequence of TlpA divided into heptad repeatportions ‘A’ through ‘M’ in the predicted coiled-coil domain, with thecoil register of heptads assigned based on consensus between previousliterature [3] and the structure prediction servers Paircoil2 [5] andLOGICOIL [6], as shown in FIG. 14. In particular, FIG. 15 shows DNAbinding domain residues 1 to 69 (SEQ ID NO:472), Heptad repeat ‘A’residues 70 to 92 (SEQ ID NO:473), Heptad repeat ‘B’ residues 93 to 108(SEQ ID NO:474), Heptad repeat ‘C’ residues 109 to 152 (SEQ ID NO: 475),Heptad repeat ‘D’ residues 155 to 178 (SEQ ID NO:476), Heptad repeat ‘E’residues 180 to 193 (SEQ ID NO:477), Heptad repeat ‘F’ residues 194 to198 (SEQ ID NO:478), Heptad repeat ‘G’ residues 199 to 221 (SEQ IDNO:479), Heptad repeat ‘H’ residues 223 to 232 (SEQ ID NO:480), Heptadrepeat ‘I’ residues 233 to 236 (SEQ ID NO:481), Heptad repeat T residues238 to 253 (SEQ ID NO: 482), Heptad repeat ‘K’ residues 254 to 262 (SEQID NO:483), Heptad repeat ‘L’ residues 263 to 278 (SEQ ID NO:484), Aminoacids between heptad repeats ‘L’ and ‘M’ (SEQ ID NO: 485), Heptad repeat‘M’ residues 309 to 361 (SEQ ID NO: 486), Amino acids after heptadrepeat ‘M’ (SEQ ID NO: 487). Amino acids that did not fit into a heptadbetween heptad repeat portions ‘A’ through ‘M’ and also following heptadrepeat portion ‘M’ are indicated with “doesn't fit to heptad”. Thepredicted DNA binding domain is also indicated (residues 1 to 69).

FIGS. 16A-16GM show ‘helical wheel’ diagrams of the predicted structureof the TlpA coiled-coil interface as viewed down the long axis of thehelix, with two TlpA proteins shown side-by-side, in a predicted dimerconfiguration of the transcriptionally repressive state. Each helicalwheel diagram (FIGS. 16A-16M show a portion of the predicted coiled-coildomain, divided into uninterrupted amino acid sequences that arepredicted to fit into the same heptad register, according to thesequences listed in FIG. 15, as follows: FIG. 16A corresponds to FIG. 15Heptad repeat ‘A’ (SEQ ID NO:473); FIG. 16B corresponds to FIG. 15Heptad repeat ‘B’ (SEQ ID NO:474); FIG. 16C corresponds to FIG. 15Heptad repeat ‘C’ (SEQ ID NO: 475); FIG. 16D corresponds to FIG. 15Heptad repeat ‘D’ (SEQ ID NO:476); FIG. 16E corresponds to FIG. 15Heptad repeat ‘E’ (SEQ ID NO:477); FIG. 16F corresponds to FIG. 15Heptad repeat ‘F’ (SEQ ID NO:478); FIG. 16G corresponds to FIG. 15Heptad repeat ‘G’ (SEQ ID NO:479); FIG. 16H corresponds to FIG. 15Heptad repeat ‘H’ (SEQ ID NO:480); FIG. 16I corresponds to FIG. 15Heptad repeat ‘I’ (SEQ ID NO:481); FIG. 16J corresponds to FIG. 15Heptad repeat ‘J’ (SEQ ID NO: 482); FIG. 16K corresponds to FIG. 15Heptad repeat ‘K’ (SEQ ID NO:483); FIG. 16L corresponds to FIG. 15Heptad repeat ‘L’ (SEQ ID NO:484); FIG. 16M corresponds to FIG. 15Heptad repeat ‘M’ (SEQ ID NO: 486). The predicted alpha-helical heptadrepeat (labeled a-b-c-d-e-f-g) is shown, connected by progressivelythinner straight lines shown in an N-terminal to C-terminal direction. Adashed line is shown between the last residue of a heptad and the firstresidue of a next heptad in a portion of the heptad repeat.Single-letter amino acid symbols shown circled at each position in aheptad. The sequence of amino acids in an N-terminal to C-terminaldirection are shown at each position of a heptad, with the first heptadin the portion of the heptad repeat shown on the line of each largecircle representing an alpha-helix, and the amino acids of consecutiveheptads are shown further out from the large circle. The coil registerwas assigned based on consensus between previous literature [3] and thestructure prediction servers Paircoil2 [5] and LOGICOIL [6]. The imageswere produced using DrawCoil 1.0 [7]. In FIGS. 16C, 16D, 16H, 16J and16M, inter-monomeric dashed lines represent predicted energeticallyfavorable ionic interactions. In FIG. 16E, lines comprised of small ‘x’indicate predictive repulsive inter-monomeric ionic interaction betweenthe indicated R and K residues. Inter-monomeric dashed lines representpredicted energetically favorable ionic interactions.

FIG. 17 shows residues 1 to 139 of the amino acid sequence of TlpAprotein (SEQ ID NO: 488), with amino acids annotated with ‘H’ on thelower row representing an amino acid that forms part of a predictedheptad repeat of a coiled-coil domain. The secondary structureprediction of TlpA was performed using the software JPred. This softwarepredicts that the DNA binding domain of TlpA consists of three alphahelices separated by short linkers.

FIGS. 18A-18B show the DNA and protein sequence of TcI. FIG. 18A showsthe DNA sequence (SEQ ID NO: 466). FIG. 18B shows the single-letteramino acid code sequence of TcI protein (SEQ ID NO: 467). Boldunderlined amino acid residues are mutated in the TcI₃₈ variant, italicunderlined residues are mutated in the TcI₄₂ variant, and the lowercaseunderlined amino acid residue is A67T that confers the originaltemperature sensitivity in the cI⁸⁵⁷ mutant (herein referred to as TcI).The ‘*’ symbol represents the stop codon at the C-terminus of theprotein.

FIGS. 19A-19B show the DNA and protein sequence of the TcI₃₈ variant.FIG. 19A shows the DNA sequence (SEQ ID NO: 468). FIG. 19B shows thesingle-letter amino acid code sequence of TcI₃₈ protein (SEQ ID NO:469). The ‘*’ symbol represents the stop codon at the C-terminus of theprotein.

FIGS. 20A-20B show the DNA and protein sequence of the TcI₄₂ variant.FIG. 20A shows the DNA sequence (SEQ ID NO: 470). FIG. 20B shows thesingle-letter amino acid code sequence of TcI₄₂ protein (SEQ ID NO:471). The ‘*’ symbol represents the C-terminus of the protein

FIG. 21 shows the amino acid sequence of TcI (SEQ ID NO: 467),highlighting the DNA binding domain (bold), the linker (italicized) andthe globular dimerization domain (underlined), based on the crystalstructure of the wild type lambda repressor (Protein Data Bank (PDB)code 3BDN) [8]. The ‘*’ symbol represents the stop codon at theC-terminus of the protein.

FIG. 22A shows a crystal structure model image depicting a dimer of TcI.The crystal structure of the wild type lambda repressor (Protein DataBank (PDB) code 3BDN) was used as the homology model [8]. The DNAbinding domain is shown in contact with a double helix DNA structurerepresenting the pr/pL binding site. FIGS. 22B-D show crystal structuresof the individual domains of dimerized TcI, wherein the DNA-bindingdomain is shown in FIG. 22B, the linker domain is shown in FIG. 22C, andthe globular dimerization domain is shown in FIG. 22D.

FIGS. 23A-D show the CD melting curves normalized and fitted accordingto Hill's equation for TlpA-CC WT, TlpA-CC-DED, DED-TlpA-CC andtropomysin.

FIG. 24 shows a Table of physicochemical properties of twenty naturallyoccurring amino acids, including side-chain class, side chain polarity,side-chain charge (at pH 7.4), hydropathy index [9], and molecularweight (g/mol).

DETAILED DESCRIPTION

Provided herein are temperature sensitive transcription factors that canbe used as thermal transcriptional bioswitches and related gene vectors,genetic circuits, methods and systems.

The term “transcription factor” as used herein indicates a proteincapable of controlling transcription of an encoded polynucleotide fromDNA to RNA by binding to a DNA regulatory sequence such as an enhancerand/or a promoter or any other DNA segment operably connected to theencoded polynucleotide.

The term “protein” as used herein indicates a polypeptide with aparticular secondary and tertiary structure that can interact withanother molecule and in particular, with other biomolecules includingother proteins, DNA, RNA, lipids, metabolites, hormones, chemokines,and/or small molecules. The term “polypeptide” as used herein indicatesan organic linear, circular, or branched polymer composed of two or moreamino acid monomers and/or analogs thereof. The term “polypeptide”includes amino acid polymers of any length including full lengthproteins and peptides, as well as analogs and fragments thereof. Apolypeptide of three or more amino acids is also called a proteinoligomer, peptide, or oligopeptide. In particular, the terms “peptide”and “oligopeptide” usually indicate a polypeptide with less than 100amino acid monomers. In particular, in a protein, the polypeptideprovides the primary structure of the protein, wherein the term “primarystructure” of a protein refers to the sequence of amino acids in thepolypeptide chain covalently linked to form the polypeptide polymer. Aprotein “sequence” indicates the order of the amino acids that form theprimary structure. Covalent bonds between amino acids within the primarystructure can include peptide bonds or disulfide bonds, and additionalbonds identifiable by a skilled person. Polypeptides in the sense of thepresent disclosure are usually composed of a linear chain of alpha-aminoacid residues covalently linked by peptide bond or a synthetic covalentlinkage. The two ends of the linear polypeptide chain encompassing theterminal residues and the adjacent segment are referred to as thecarboxyl terminus (C-terminus) and the amino terminus (N-terminus) basedon the nature of the free group on each extremity. Unless otherwiseindicated, counting of residues in a polypeptide is performed from theN-terminal end (NH₂-group), which is the end where the amino group isnot involved in a peptide bond to the C-terminal end (—COOH group) whichis the end where a COOH group is not involved in a peptide bond.Proteins and polypeptides can be identified by x-ray crystallography,direct sequencing, immuno precipitation, and a variety of other methodsas understood by a person skilled in the art. Proteins can be providedin vitro or in vivo by several methods identifiable by a skilled person.In some instances where the proteins are synthetic proteins in at leasta portion of the polymer two or more amino acid monomers and/or analogsthereof are joined through chemically-mediated condensation of anorganic acid (—COOH) and an amine (—NH₂) to form an amide bond or a“peptide” bond.

As used herein the term “amino acid”, “amino acidic monomer”, or “aminoacid residue” refers to organic compounds composed of amine andcarboxylic acid functional groups, along with a side-chain specific toeach amino acid. In particular, alpha- or α-amino acid refers to organiccompounds composed of amine (—NH₂) and carboxylic acid (—COOH), and aside-chain specific to each amino acid connected to an alpha carbon.Different amino acids have different side chains and have distinctivecharacteristics, such as charge, polarity, aromaticity, reductionpotential, hydrophobicity, and pKa. Amino acids can be covalently linkedto form a polymer through peptide bonds by reactions between the aminegroup of a first amino acid and the carboxylic acid group of a secondamino acid. Amino acid in the sense of the disclosure refers to any ofthe twenty naturally occurring amino acids, non-natural amino acids, andincludes both D an L optical isomers.

The term “non-natural amino acids” or “artificial amino acids” indicatenot naturally encoded or found in the genetic code of any organisms andtypically comprise non-proteinogenic amino acids that either occurnaturally or are chemically synthesized. Accordingly, non-natural aminoacids comprise molecules that can be coupled together using standardamino acid coupling chemistry, and that have molecular structures thatdo not resemble the naturally occurring amino acids. Exemplarynon-natural amino acids comprise e.g., α,α′-dialkyl-amino acids such asamino isobutyric acid (Aib) or cyclopentyl glycine and analogs ofnaturally occurring amino acids. The term “amino acid analog” refers toan amino acid in which one or more individual atoms have been replaced,either with a different atom, isotope, or with a different functionalgroup but is otherwise identical to original amino acid from which theanalog is derived [10].

Transcription factors in the sense of the disclosure are proteins thatcontrol expression of an encoded polynucleotide by binding DNAregulatory sequences alone or in combination with one or moretranscription co-factors.

The term “DNA regulatory sequence” as used herein indicates any DNAsegment operably connected to an encoded polynucleotide. The terms“operably connected” or “operably linked” between two elements refer toa functional linkage between the two elements, so that functionalitiesof one element is controlled by the other element. In DNA regulatorysequence a conformation of the DNA segment triggers transcription of theencoded polynucleotide. Operably linked DNA regulatory sequences andencoded polynucleotide can be contiguous or non-contiguous and cancomprise polynucleotides in a same or different reading frame. In anembodiment, each of the operably linked polynucleotide can be comprisedwithin a cassette. The cassette can additionally contain at least oneadditional polynucleotide to be co-expressed with the encodedpolynucleotide (e.g. a selectable marker gene). One or more additionalgenes can also be provided on multiple expression cassettes that canfurther comprise a plurality of restriction sites and/or recombinationsites for insertion of other polynucleotides.

Transcription co-factors in the sense of the disclosure refer toproteins polynucleotides or portions therefore that are configured tocontrol transcription of an encoded polynucleotide in combination withtranscription factors, e.g. by binding the transcription factor to formtranscription complex. Examples of transcription co-factors that cancontrol transcription in combination with transcription factors in thesense of the disclosure comprise transcriptional or translationalcontrolling factors such CRISPR-CAS systems, siRNA, riboregulators,transcriptional RNA based activators and repressors [11], and otherfactors identifiable by a skilled person. Transcription factors andtranscription cofactor of the disclosure that can be naturally derived,purely synthetically derived, or synthetically derived from naturalsystems.

Transcription factors in the sense of the disclosure comprisetranscription repression factor (also referred to as “repressor”) and atranscription activation factor (also referred to as “activator”). Thetranscription repression factor binds to DNA regulatory sequence torepress the transcription of an encoded polynucleotide, thereby reducingthe expression level of the encoded polynucleotide. The transcriptionactivation factor binds to DNA regulatory sequence to promote thetranscription of an encoded polynucleotide, thereby increasing theexpression level of the encoded polynucleotide. In particular, atranscription regulatory factor has typically at least one DNA-bindingdomain that can bind to a DNA regulatory sequence such as an enhancer ora promoter. Some transcription factors bind to a DNA promoter sequencenear the transcription start site to form the transcription initiationcomplex. Other transcription factors bind to other regulatory sequences,such as enhancer sequences, and can either stimulate or represstranscription of the related gene. Examples of transcription repressionfactors include TlpA, TetR, LacI, LambdaCI, PhlF, SrpR, QacI, BetR,LmrA, AmeR, LitR, met, and other identifiable by a skilled person, aswell as homologues of known repression factors, that function in bothprokarayotic and eukarayotic systems. Examples of transcriptionactivation factors include AraC, LasR, LuxR, IpgC, MxiE, Gal4, GCN4, GR,SP1, CREB, etc as well as homologues of known activation factors, thatfunction in both prokarayotic and eukarayotic systems.

“Temperature sensitive transcription factors” “thermal transcriptionalbioswitches” or “transcriptional bioswitches” in the sense of thedisclosure herein also indicated as “transcriptional bioswitches” aretranscription factors that have a DNA-bound state or conformation inwhich the transcription factor is specifically bound to a correspondingDNA regulatory sequence through a DNA binding domain, and a DNA unboundstate or conformation in which the transcription factor is not bound toa corresponding DNA regulatory sequence.

The term “corresponding” as used herein in connection with molecules orresidues within a molecule, indicates the ability of the referenceditems to react to one another. Thus a DNA regulatory sequence and a DNAbinding domain that can react to one another are indicated as“corresponding” Similarly, amino acid residues within a protein or indifferent protein and that can react with one another can be referred toas corresponding amino acid residues. Also structural motifs within asame or in different molecules that can react and in particular bind oneto another can also be referred as “corresponding”.

In a temperature sensing transcription factor, the factor can convertfrom a DNA-bound state to a DNA-unbound state with reference tocorresponding DNA regulatory sequence at a bioswitch temperature Tbs.

In particular, temperature sensitive factors in the sense of thedisclosure comprise transcriptional bioswitch dimers formed by twomonomer proteins. The term “dimer” as used herein indicates amacromolecular complex formed by two polymers and in particular twopolypeptides. In a dimer the two protein monomers bind to one anotherthrough covalent and/or non-covalent interactions as will be understoodby a skilled person. Examples of non-covalent interactions compriseionic bonds, Van der Waals interactions, polar interactions, saltbridges, coulombic attraction, coulombic repulsion, hydrophobicinteraction, and others identifiable by a skilled person. An example ofa non-covalently bound protein dimer is the enzyme reversetranscriptase. Examples of covalent interactions comprise any chemicalbond that involves the sharing of electron pairs between such asdisulfide bridges.

Dimers in the sense of the disclosure can be homodimers andheterodimers. The term “homodimer” means a dimer consisting of twomonomers with identical polymer sequence, and in particular twopolypeptide or protein monomers with identical amino acid sequence.Examples of protein homodimers include the enzyme cyclooxygenase (COX),the methionine repressor MetJ, TlpA, the lambda repressor cI, and othersidentifiable by a skilled person. The term “heterodimer” means a dimerof two monomers with non-identical polymer sequence, and in particulartwo polypeptide or protein monomers with non-identical amino acidsequence. Examples of protein heterodimers include the enzyme reversetranscriptase and others identifiable by a skilled person.

In some embodiments herein described, a protein dimer forming atemperature sensitive transcription factor herein described comprises aDNA binding domain and a temperature-sensing domain.

The term “domain” as related to the protein indicates any continuouspart of a protein sequence from single amino acid up to the full proteinassociated to an identifiable structure within the protein. An“identifiable structure” in the sense of the disclosure indicates aspatial arrangement of the primary structure or portions thereof whichcan be detected by techniques such as crystallography, hydrophobicityanalysis or additional techniques known by a skilled person. In manyinstances, a protein domain comprises one or more secondary structuresof the protein, which together form a tertiary or quaternary structureof a protein.

The “secondary structure” of a protein refers to local sub-structureswith a repeating geometry identifiable within crystal structure of theprotein, circular dichroism or by additional techniques identifiable bya skilled person. In some instances, a secondary structure of a proteincan be identified by the patterns of hydrogen bonds between backboneamino and carboxyl groups. Secondary structures can also be definedbased on a regular, repeating, geometry, being constrained toapproximate values of the dihedral angles w and y of the amino acids inthe secondary structure unit on the Ramachandran plot. Two main types ofsecondary structure are the alpha helix and the beta strand or betasheets as will be identifiable by a skilled person. Both the alpha helixand the beta sheet represent a way of establishing non-covalent hydrogenbonds between constituents of the peptide backbone, thus formingsecondary structural features. Secondary structure formation can bepromoted by formation of hydrogen bonds between backbone atoms. Aminoacids that can minimize formation of a secondary structure bydestabilizing the structure of the hydrogen bonding interactions arereferred to as secondary structure breakers. Amino acids that canpromote formation of a secondary structure by stabilizing formation ofhydrogen bonding interactions are referred to as structure makers.

The term “tertiary structure” refers to the three-dimensional structureof a protein, stabilized by non-covalent interactions among non-adjacentsegments of the protein and optionally by one or more additionalcompounds or ions interacting through covalent or non-covalentinteractions with one or more segments of the proteins. Exemplarynon-covalent interactions stabilizing the three dimensional structure ofthe proteins comprise non-specific hydrophobic interactions, burial ofhydrophobic residues from water, specific tertiary interactions, such assalt bridges, hydrogen bonds, the tight packing of side chains,chelation and disulfide bonds and additional interactions identifiableby a skilled person. Exemplary covalent interactions among compounds orions and segments of the protein comprise N-linked glycosylation,cytochrome C haem attachment and additional interaction identifiable bya skilled person.

The term ““quaternary structure” when referred to a complex refers tothe three dimensional structure of a protein complex, also called amultimer, stabilized by non-transitory interactions between the two ormore proteins forming the complex. Accordingly, the quaternary structurecan be stabilized by some of the same types of non-covalent and covalentinteractions as the tertiary structure as will be understood by askilled person. Multimers made up of identical subunits are referred towith a prefix of “homo-” (e.g. a homotetramer) and those made up ofdifferent subunits are referred to with a prefix of “hetero-”, forexample, a heterotetramer, such as the two alpha and two beta chains ofhemoglobin. “Non-transitory interactions” as used herein indicatesinteractions between proteins or related segments that are detectable bylaboratory techniques such as immunoprecipitation, crosslinking andForster Resonance Energy Transfer (FRET) measurements, crystallography,Nuclear Magnetic Resonance (NMR) and additional techniques identifiableby a skilled person.

Detection of three-dimensional secondary, tertiary, or quaternaryprotein structure can be performed using techniques such as x-raycrystallography, NMR spectroscopy, dual polarization interferometryamong others known to a skilled person. Using such techniques, theposition of structural features comprising alpha-helices, beta strands,turns, beta bridges, bends, loops, coils, coiled coils, and othersidentifiable by a skilled person within the N-terminal to C-terminalprimary sequence of a protein can be detected. In particular, detectionof repeated motifs in any one of SEQ ID NO:1 to SEQ ID NO: 456 in acoiled coil domain can be performed using structure prediction serversCOILS [4], Paircoil 2 [5], LOGICOIL [6] and JPred. Structure ofpolypeptides and proteins can also be obtained from publicly availablesources such as Protein Data Bank [12] and others known to a skilledperson.

In embodiments herein described, the term “DNA binding domain” refers toa protein domain that contains at least one structural motif configuredto recognize and bind double- or single-stranded DNA, wherein the term“motif” refers to a supersecondary structure that appears in multipleprotein, and in particular a three-dimensional protein structure ofseveral adjacent elements of a secondary structure that is smaller thana protein domain or a subunit. DNA-binding domains can be part of alarger protein consisting of further protein domains with differingfunctions including the function of regulating the activity of theDNA-binding domain. The function of DNA binding can be either structuralor involve transcription regulation, or both. Many proteins involved inthe regulation of gene expression contain DNA-binding domains as will beunderstood by a skilled person. Such proteins include transcriptionfactors, or transcriptional repressors, among others recognizable by askilled person.

A DNA-binding domain in the sense of the disclosure can recognize andbind DNA in a DNA sequence-specific or non-sequence-specific manner,which involves molecular complementarity between protein and DNA. Thewording “specific” “specifically” or “specificity” as used herein withreference to the binding of a first molecule to second molecule refersto the recognition, contact and formation of a stable complex betweenthe first molecule and the second molecule, together with substantiallyless to no recognition, contact and formation of a stable complexbetween each of the first molecule and the second molecule with othermolecules that may be present. Exemplary specific bindings areantibody-antigen interaction, cellular receptor-ligand interactions,polynucleotide hybridization, enzyme substrate interactions etc. Theterm “specific” as used herein with reference to a molecular componentof a complex, refers to the unique association of that component to thespecific complex which the component is part of. The term “specific” asused herein with reference to a sequence of a polynucleotide refers tothe unique association of the sequence with a single polynucleotidewhich is the complementary sequence. By “stable complex” is meant acomplex that is detectable and does not require any arbitrary level ofstability, although greater stability is generally preferred

In embodiments herein described, a DNA-binding domain of a protein canperform DNA recognition and DNA specific binding for example at themajor or minor groove of DNA, or at the sugar-phosphate DNA backbone.DNA-binding domains can recognize specific DNA sequences, such as someDNA-binding domains of transcription factors that activate specificgenes, or some DNA-binding domains of transcriptional repressors thatrepress the transcription of specific genes. Another example is that ofenzymes that modify DNA at specific sites, such as restriction enzymes.In particular, the DNA binding domain adopts correctly-orientedalignment of its constituent sub-components to effectively interact withDNA.

The specificity of DNA-binding proteins can be detected using manybiochemical and biophysical techniques, such as gel electrophoresis,analytical ultracentrifugation, calorimetry, DNA mutation, proteinstructure mutation or modification, nuclear magnetic resonance, x-raycrystallography, surface plasmon resonance, electron paramagneticresonance, cross-linking and microscale thermophoresis (MST), amongothers recognizable by a skilled person.

In some embodiments herein described where the temperature sensitivetranscription factor is a dimer, DNA binding domains of the temperaturesensitive transcription factors can be configured to bind with a DNAregulatory sequence upon dimerization of the protein monomers, andtherefore be dimerization dependent. The term “dimerization” refers tothe process of forming a dimer of two monomers, for example two proteinmonomers. In particular, dimerization dependent DNA binding domains areconfigured so that dimerization of the monomer components strengthensthe interactions of the domain with a corresponding DNA regulatorysequence, rendering the formation or dissociation of the dimers anintrinsic part of the regulatory mechanisms. Examples ofdimerization-dependent DNA binding domains include helix-turn-helixDNA-binding domains or proteins such as tryptophan repressor, lambdaCro, lambda repressor fragment, catabolite gene activator protein (CAP)fragment. In particular, dimerization dependent DNA binding domains canbind to DNA sequences that are composed of two very similar“half-sites,” typically also arranged symmetrically. This arrangementallows each protein monomer of the to make a nearly identical set ofcontacts and enormously increases the binding affinity.

In some embodiments, dimerization dependent DNA binding domains areselected from helix-loop-helix, helix-turn-helix, zinc finger, leucinezipper, winged helix, winged helix turn helix, helix loop helix,HMG-box, Wor3 domain, OB-fold domain, immunoglobulin fold, B3 domain,TAL effector DNA-binding domain, and others recognizable by a skilledperson.

In some embodiments, the dimerization-dependent DNA-binding domainsherein described comprise a plurality of helical peptide segments eachhaving a primary structure configured to form an alpha helix secondarystructure. The term “alpha helix” or “α-helix” indicates aright-hand-coiled or spiral conformation (helix) of a polypeptide inwhich every backbone N—H group donates a hydrogen bond to the backboneC═O group of the amino acid four residues earlier facilitating hydrogenbonding. The alpha helix is a common secondary structure of proteins andis also sometimes called a classic Pauling-Corey-Branson alpha helix or3.6₁₃-helix, the latter indication denoting the number of residues perhelical turn, and 13 atoms being involved in the ring formed by thehydrogen bond.

Exemplary dimerization dependent DNA binding domains comprising aplurality of helical peptide segments comprise Helix-turn-helix andbasic helix-loop-helix, zinc finger, basic leucine zipper, wingedhelix-turn-helix, HMG box, Wor3, O-B fold, immunoglobulin domain, B3 DNAbinding domain and Tal effector

“Helix-turn-helix” indicates a motif composed of two a helices joined bya short strand of amino acids. In particular, the two a helices, thefirst one occupying the N-terminal end of the motif, and the second oneat the C-terminus, perform recognition and binding to DNA ofhelix-turn-helix proteins. In most cases, such as in the Cro repressor,the second helix contributes most to DNA recognition, and hence it isoften called the “recognition helix”. It binds to the major groove ofDNA through a series of hydrogen bonds and various Van der Waalsinteractions with exposed bases. The first a helix stabilizes theinteraction between protein and DNA, but typically does not play aparticularly strong role in its recognition. The recognition helix andits preceding helix always have the same relative orientation.Helix-turn-helix motifs can be classified based on their structure andthe spatial arrangement of their helices, for example di-helical,tri-helical, tetra-helical, or winged helix-turn-helix, among othersidentifiable by a skilled person. For example, helix-turn-helix motifare found in transcription regulatory proteins from bacteriophage lambdaand Escherichia coli: Cro, CAP, and λ repressor, which share a common20-25 amino acid sequence that facilitates DNA recognition. Otherhelix-turn-helix-containing proteins are recognizable by a skilledperson.

“Basic helix-loop-helix (“bHLH”)” indicates a motif formed by twoα-helices connected by a loop. In general, transcription factorsincluding bHLH domain are dimeric, each with one helix containing basicamino acid residues that facilitate DNA binding. In general, in a bHLHone helix is smaller, and, due to the flexibility of the loop, allowsdimerization by folding and packing against another helix. In a bHLH DNAbinding domain the larger helix typically contains the DNA-bindingregions. bHLH proteins typically bind to a consensus sequence called anE-box, CANNTG. [6] The canonical E-box is CACGTG (palindromic), howeversome basic helix-loop-helix transcription factors, notably those of thebHLH-PAS family, bind to related non-palindromic sequences, which aresimilar to the E-box. Examples of transcription factors containing abHLH include AhR, Beta2/NeuroD1, BMAL-1-CLOCK, C-Myc, N-Myc, MyoD, Myf5,Pho4, HIF, ICE1, NPAS1, NPAS3, MOPS, Scl, also known as Tall, proneuralbHLH genes like p-CaMKII, and pSer(336)NeuroD, Scleraxis, Neurogenins,MAX, OLIG1, OLIG2, and TCF4 (Transcription Factor 4), among otheridentifiable by a skilled person

The term “zinc finger” indicates a motif that is characterized by thecoordination of one or more zinc ions in order to stabilize the fold.Proteins that contain zinc fingers (zinc finger proteins) are classifiedinto several different structural families. There are a number of typesof zinc fingers, each with a unique three-dimensional architecture. Aparticular zinc finger protein's class is determined by thisthree-dimensional structure, but it can also be recognized based on theprimary structure of the protein or the identity of the ligandscoordinating the zinc ion. In general a zinc finger can comprise between23 and 28 amino acids long and is stabilized by coordinating zinc ionswith regularly spaced zinc-coordinating residues (either histidines orcysteines). The most common class of zinc finger (Cys2His2) coordinatesa single zinc ion and consists of a recognition helix and a 2-strandbeta-sheet. Other classes of zinc finger domain are identifiable by askilled person. In transcription factors these domains are often foundin arrays (usually separated by short linker sequences) and adjacentfingers are spaced at 3 basepair intervals when bound to DNA as will beunderstood by a skilled person.

The term “basic leucine zipper (bZIP)” indicates a motif that containsan alpha helix with a leucine at every 7th amino acid. If two suchhelices find one another, the leucines can interact as the teeth in azipper, allowing dimerization of two proteins. When binding to the DNA,basic amino acid residues bind to the sugar-phosphate backbone while thehelices sit in the major grooves.

The term “winged helix (WH)” indicates a motif comprises four helicesand a two-strand beta-sheet and is typically about 110 amino acids.Winged helix motifs can be found in transcription factors e.g. thetranscription factors classified into 19 families called FoxA-FoxS, aswill be understood by a skilled person.

The term “winged helix-turn-helix (wHTH)” indicates a motif formed by awinged helix-turn-helix DNA-binding motif, where the “wings”, or loops,are small beta-sheets. Typically a wHTH is a motif 85-90 amino acidslong formed by a 3-helical bundle (H1, H2, H3) and three beta-sheets(S1, S2, S3) and two wings (W1, W2), arranged in the orderH1-S1-H2-H3-S2-W1-S3-W2. In wHTH, The DNA-recognition helix makessequence-specific DNA contacts with the major groove of DNA, while thewings make different DNA contacts, often with the minor groove or thebackbone of DNA. Several winged-helix proteins display an exposed patchof hydrophobic residues thought to mediate protein-protein interactions.

The term “HMG-box” indicates a motif formed by three alpha helicesseparated by loops. HMG-box can be found in high mobility group proteinswhich are involved in a variety of DNA-dependent processes such asreplication and transcription. HMG-box motifs also alter the flexibilityof the DNA by inducing bends as will be understood by a skilled person.

The term “Wor3” indicates a motif named after the White-Opaque Regulator3 (Wor3) in Candida albicans which is described in Lohse et al 2013 [13]which is capable of DNA specific binding.

The term “OB-fold indicates a small structural motif originally namedfor its oligonucleotide/oligosaccharide binding properties described inFlynn et al 2010 [14]. OB-fold can range between 70 and 150 amino acidsin length and bind single-stranded DNA, and therefore can be included inDNA binding domain in single-stranded binding proteins. OB-fold proteinshave been identified as critical for DNA replication, DNA recombination,DNA repair, transcription, translation, cold shock response, andtelomere maintenance.

The term “immunoglobulin” refers to a motif consisting of a beta-sheetstructure with large connecting loops, which serve to recognize eitherDNA major grooves or antigens. Usually found in immunoglobulin proteins,immunoglobulin motifs are also present in Stat proteins of the cytokinepathway as will be understood by a skilled person.

The term “B3” indicates a motif including seven beta sheets and twoalpha helices, which form a DNA-binding pseudobarrel protein fold. B3motif typically consists of 100-120 residues and is naturally foundexclusively in transcription factors from higher plants and restrictionendonucleases EcoRII and BfiI and

The term “TAL effectors” indicates a motif containing between 1.5 and33.5 repeats that are usually 34 residues in length (the C-terminalrepeat is generally shorter and referred to as a “half repeat”). Atypical repeat sequence is LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG, (SEQ IDNO: 458) but the residues at the 12th and 13th positions arehypervariable (these two amino acids are also known as the repeatvariable diresidue or RVD). Typically TAL effector can comprise acentral region of tandem 33-35 residue repeats and each repeat regionencodes a single DNA base in the TAL effector's binding site. Within therepeat residue 13 directly contacts the DNA base, determining sequencespecificity, while other positions make contacts with the DNA backbone,stabilizing the DNA-binding interaction. Each repeat within the arraytypically takes the form of paired alpha-helices, while the whole repeatarray forms a right-handed superhelix, wrapping around the DNA-doublehelix. Tal effectors are naturally found in bacterial plant pathogens ofthe genus Xanthomonas among others as will be understood by a skilledperson.

In some embodiments, of the transcriptional bioswitch dimers hereindescribed, in each monomer protein the C-terminus of the dimerizationdependent DNA binding domain is covalently attached to the N-terminus ofthe temperature sensitive domain.

The term “attach” or “attached” as used herein, refers to connecting oruniting by a bond, link, force or tie in order to keep two or morecomponents together, which encompasses either direct or indirectattachment where, for example, a first molecule is directly bound to asecond molecule or material, or one or more intermediate molecules aredisposed between the first molecule and the second molecule or material.In particular in some embodiments, the dimerization dependent DNAbinding domain and the temperature sensitive domain can be directlyfused to each other, or fused to an interceding linker. The linker canbe short (1-5 residues), intermediate (5-10), or long (<10) and can berigid or flexible Additional linkers are identifiable by a skilledperson upon reading of the present disclosure [15]

The term “temperature-sensing domain” refers to a protein or a portionthereof having a sequence configured to provide structural lability inresponse to temperature changes.

In some embodiments, the temperature sensitive transcription factor is acoiled coil temperature interaction domain, and the temperature-sensingdomain is a coiled coil temperature sensing domain comprisingtemperature sensing supercoiled motif of alpha-helical secondarystructures. In particular, the term “coiled coil” indicates a structuralmotif in a protein in which two to seven alpha-helices are coiledtogether like the strands of a rope and interact with coiled coilstructural motifs in one or more other proteins. Dimers and trimers arethe most common types. Coiled coils usually contain a repeated pattern,“hxxhcxc”, of hydrophobic (h)- and charged or polar (c) amino-acidresidues, referred to as a heptad repeat. The positions in the heptadrepeat can be labeled “abcdefg”, according to a register where “a” and“d” are generally hydrophobic positions, often being occupied byisoleucine, leucine, or valine.

The term “register” as used herein in relation to a heptad repeatindicates the sequence of the positions a, b, c, d, e, f, g within theheptad repeat in an alpha-helical coiled coil. In particular a registerindicates a series of consecutive positions among the possibleconsecutive a, b, c, d, e, f and g positions starting at any one ofpositions a, b, c, d, e, f, or g and can be interrupted by variation ofthe sequence such as deletion or insertions. A heptad coil register canbe assigned based on consensus between previous literature [3] andstructure prediction servers including COILS [4], Paircoil 2 [5],LOGICOIL [6], and Jpred. Folding a sequence with this repeating patterninto an alpha-helical secondary structure causes the generallyhydrophobic “a” and “d” residues to be presented as a stripe that coilsaround the helix, forming an amphipathic structure as will be understoodby a skilled person.

In a coiled coil temperature sensing domain, alpha helices of the coiledcoil motif form a tertiary structure in a water-filled environment suchas the cytoplasm, and in particular the hydrophobic strands are wrappedagainst each other and are sandwiched between the hydrophilic aminoacids. The alpha-helices can be parallel or anti-parallel, and can adopteither a left-handed or right-handed coiled coil. Coiled coils can bedepicted using a ‘helical wheel’ diagram, in which the coiled coils areviewed down the axis of the alpha-helices from N-terminus to C-terminussuch as the exemplary structure schematically illustrated in FIGS.16A-16M with reference to the coiled coil domain of TlpA, which show aseries of helical wheel representations of the homodimeric coiled-coil,with each monomer coil made up of heptad repeats.

In the coiled coil transcriptional bioswitch dimers herein described,the temperature sensitive domain of each monomer protein has atemperature sensitive amino acid amino acid sequence having a lengthfrom 14 to 3200 amino acid residues, and a sequence

-   -   [X₁ X₂ X₃ X₄ X₅ X₆ X₇]_(a)        wherein X₁ is a hydrophobic amino acid, X₂ is a polar or charged        amino acid, X₃ is a polar or charged amino acid, X₄ is a        hydrophobic amino acid, X₅ is a polar or charged amino acid, X₆        is a polar or charged amino acid, and X₇ is a polar or charged        amino acid and n can be any integer between 2 and 457 (SEQ ID        NO: 1 to SEQ ID NO: 456).

The term “polar” as used herein means a molecule, and in particular anamino acid, having a side chain that includes a functional group thathave a dipole moment greater than C—H bond. Exemplary polar functionalgroups include carboxylic acid, ester, amide, nitrile, aldehyde, ketone,hydroxyl group, amino group, and mercapto group.

Exemplary polar amino acids include serine, threonine, asparagine,glutamine, histidine and tyrosine. The nature of side chain of an aminoacid residue in a protein or peptide affects the interaction with waterin an aqueous environment such as those found in cells.

Hydrophobicity refers to the property of thermodynamically unfavorableinteraction with water. In contrast, hydrophilicity refers to theproperty of thermodynamically favorable interaction with water.

Thus, hydrophobic effect represents the tendency of water to excludenon-polar molecules. The effect originates from the disruption ofhydrogen bonds between water molecules. Hydrophobic molecules tend to benonpolar and, thus, prefer other neutral molecules and nonpolarsolvents. Because water molecules are polar, hydrophobic molecules donot dissolve well among them. Hydrophobic molecules in water oftencluster together.

There are different hydrophobicity scales (or alternatively, hydropathyindices) of amino acid residues, based on measurement of the level ofdisruption of hydrogen bonds between water molecules, such as that ofKyte and Doolittle [9] as shown in FIG. 24.

Exemplary hydrophobic amino acids include alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan, cysteine and methionine.

Hydrophobicity scales or hydropathy indices are values that can be usedto define relative hydrophobicity or conversely relative polarity(hydrophilicity) of amino acid residues, wherein, the more positive thevalue, the more hydrophobic the amino acid, and conversely the morenegative the value, the more polar the amino acid.

Techniques of measuring amino acid hydrophobicity or polarity comprisewet lab methods such as partitioning between two immiscible phases orreverse phase liquid chromatographic methods, or computer-based methodssuch as calculation of solvent accessible surface area.

Generally, non-polar, hydrophobic amino acids can be considered to havea hydropathy index above zero, with the most hydrophobic, nonpolar aminoacids having a Kyte and Doolittle (1982) hydropathy index value above 2,while polar, hydrophilic amino acids have a Kyte and Doolittle (1982)hydropathy index value below zero, with the most polar amino acidshaving a value below −2. For example, the polar amino acid arginine hasa hydropathy index of −4.5, whereas the hydrophobic amino acid leucinehas a hydropathy index of 3.8, and the amino acid isoleucine has ahydropathy index of 4.5, thus isoleucine can be considered morehydrophobic than leucine.

Thus, for example, replacement of a hydrophobic amino acid with ahydrophilic amino acid in a polypeptide or protein can change thestructure of a polypeptide or protein, or protein-protein interactions,and related functional characteristics of the polypeptide or protein.

The term “charged” as used herein means a molecule, and in particular anamino acid that has an ionically charged side chain, in particular atphysiological pH of an intracellular cellular environment, as understoodby a skilled person. The α-carboxylic acid group of amino acids is aweak acid, meaning that it releases a proton at moderate pH values. Inother words, carboxylic acid groups (—CO₂H) can be deprotonated tobecome negative carboxylates (—CO₂—). The negatively charged carboxylateion predominates at pH values greater than the pKa of the carboxylicacid group.

In a complementary fashion, the α-amine of amino acids is a weak base,meaning that it accepts a proton at moderate pH values. In other words,α-amino groups (NH₂—) can be protonated to become positive α-ammoniumgroups (+NH₃—). The positively charged α-ammonium group predominates atpH values less than the pKa of the α-ammonium group.

Among the twenty common natural amino acids, five have a side chainwhich can be charged. At pH=7.4, two are negatively charged: asparticacid (Asp, D) and glutamic acid (Glu, E) (acidic side chains), and threeare positive charged: lysine (Lys, K), arginine (Arg, R) and histidine(His, H) (basic side chains).

Among the twenty common natural amino acids, five have a side chainwhich can be charged. At pH=7.4, two are negatively charged: asparticacid (Asp, D) and glutamic acid (Glu, E) (acidic side chains), and threeare positive charged: lysine (Lys, K), arginine (Arg, R) and histidine(His, H) (basic side chains).

Hydrophilic amino acids are amino acid that are considered to be solublein water and have polar side chains, e.g. comprising —COOH, —OH, —NH₃,groups and other groups identifiable by a skilled person. Exemplaryhydrophilic amino acid comprise polar or charged amino acid (e.g. polarnaturally occurring amino acid serine (Ser), threonine (Thr), asparagine(Asn), glutamine (Gln), and tyrosine (Tyr); and charged naturallyoccurring amino acid such as lysine (Lys) (+), arginine (Arg) (+),aspartate (Asp) (−) and glutamate (Glu) (−). Hydrophobic amino acids areamino acids that have aliphatic or saturated hydrocarbon side chains(e.g natural occurring glycine (Gly), alanine (Ala), valine (Val),leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe),methionine (Met), and tryptophan (Trp). Polar amino acid can also beinvolved in hydrogen bond.

In some embodiments, the temperature sensitive amino acid amino acidsequence can have a length of 100 to 500 amino acid residues and n=anyinteger between 12 and 71, or 250 to 350 amino acid residues, and n=anyinteger between 32 and 50.

In the coiled coil transcriptional bioswitch dimers, the coiled coiltemperature sensing amino acid sequence of each monomer protein cancomprise one or more insertions, deletions or replacement. The term“insertion” as used herein means an introduction of one or more aminoacids between any two structural features or an introduction of one ormore amino acids within one or more structural features in an amino acidsequence. The term “deletion” as used herein means removal of one ormore amino acids comprising one or more structural features in an aminoacid sequence. The term “replacement” as used herein means substitutionof one or more amino acids comprising one or more structural features inan amino acid sequence.

In embodiments, herein described the coiled coil temperature sensingdomain of SEQ ID NO: 1 to 456 can include one or more insertions,deletions and/or replacements within a percent variation from 0% to 20%along the total length of the sequences SEQ ID NO: 1 to SEQ ID NO: 456.

The term “percent variation” or “percentage variation” as used hereinmeans the difference between two amino acid residue sequences, expressedas a percentage, wherein the difference between two amino acid sequencesis measured by a process that comprises the steps of aligning the twoamino acid sequences, then detecting one or more differences between thealigned sequences, and calculating the total number of differencesdivided by the total number of aligned amino acids in each amino acidsequence, including gaps with the result expressed as a percentage. Theterm “alignment” as used herein means aligning the positions ofstructural features of statistically significant structural similaritybetween two amino acid sequences, where “statistically significantstructural similarity” means greater than 95% probability that twostructural features are structurally homologous, for example,alpha-helix. The term ‘difference” indicates mismatches in the positionof structural features in the position of structural features in the twoamino acid sequences, whereby each amino acid that comprises part of amismatched structural feature is counted as one difference between thetwo aligned amino acid sequences. Mismatches between aligned sequencescan comprise an insertion, a deletion, and/or a replacement of one ormore structural features in one amino acid sequence compared to theother aligned amino acid sequence as would be understood by a skilledperson. Several publicly available online servers can be used to detectprotein structure alignment and calculate percent variation, such asFATCAT [16], SuperPose [17], iPBA [18], MAPSCI [19], and others known toa person skilled in the art.

In the coiled coil temperature sensing domain, each monomer protein ofthe domain has a resulting temperature sensing amino acid sequencepossibly variated in which the amino acid residues X₁ to X₇ are arrangedin the temperature sensing amino acid sequence to form at least twocomplete consecutive uninterrupted heptad repeats without modificationof any of positions a, b, c, d, e, f, or g, and possibly additionalinterrupted or uninterrupted heptad repeats each having a register inwhich up to 5 consecutive amino acid residues are optionally missing inview of possible insertions, deletions and/or replacements on SEQ ID NO:1 to SEQ ID NO: 456 within the 0% to 20% variation range to have a totalof 2 to 457 consecutive uninterrupted heptad repeats in the temperaturesensing amino acid sequence.

Accordingly, in a temperature sensitive amino acid sequence amino acidresidues X₁ to X₇ can be arranged in heptad repeats each with a registerthat begins with either a, b, c, d, e, f or g. Accordingly, anuninterrupted series of heptad repeats without modification of any oneof the a to g positions beginning at position a has a heptad repeatregister of [a, b, c, d, e, f, g]_(n); beginning at position b has aheptad repeat register of [b, c, d, e, f, g, a]_(n); beginning atposition c has a heptad repeat register of [c, d, e, f, g, a, b]_(n);beginning at position d has a heptad repeat register of [d, e, f, g, a,b, c]_(n); beginning at position e has a heptad repeat register of [e,f, g, a, b, c, d]_(n); beginning at position f has a heptad repeatregister of [f, g, a, b, c, d, e]_(n); and beginning at position g has aheptad repeat register of [g, a, b, c, d, e, f]_(n). Heptad repeats in aregister can have up to 5 consecutive amino acid residues missing inview of possible deletion or insertion in the sequence within the 0% to20% percent variation range.

Detection of heptad repeats within a coiled coil amino acid sequence canbe performed using structure prediction servers COILS [4], Paircoil 2[5], LOGICOIL [6], among others identifiable by a skilled person. Inparticular, COILS [4] detects predicted heptad repeats within an aminoacid sequence and provides a probability score for each amino acidrelative to the position a, b, c, d, e, f, or g within the heptad.

In some embodiments, variation in the temperature sensing amino acidsequence SEQ ID NO: 1 to SEQ ID NO: 456 can result in having a total of12 to 71 consecutive uninterrupted heptad repeats in the temperaturesensing amino acid sequence within the 0% to 20% variation range,

In some embodiments, variation in the temperature sensing amino acidsequence SEQ ID NO: 1 to SEQ ID NO: 456 can result in having a total of32 to 50 consecutive uninterrupted heptad repeats in the temperaturesensing amino acid sequence within the 0% to 20% variation range

In some embodiments, variation in the temperature sensing amino acidsequence SEQ ID NO: 1 to SEQ ID NO: 456 can result in having heptadrepeats interrupted by one or more insertions distributed unevenlythroughout the system.

In some embodiments, a Minimum and Maximum number of amino acidsresidues can be inserted in the coiled coil temperature sensitive aminoacid sequence, wherein—Minimum=1. Maximum=maximum number of aminoacids−(minimum number of heptad repeats*7) to provide the temperaturesensitive amino acid sequence with breaks that can be distributedunevenly throughout the coil, with long stretches of heptads and shortpuncta of heptads. Accordingly in some embodiments the number ofuninterrupted heptad repeats in the coiled coil temperature sensitiveamino acid sequence can be 2 to 30, in some embodiments the number ofuninterrupted heptad repeats in the coiled coil temperature sensitiveamino acid sequence can be 5-20 based on the consensus coiled coilprediction for TlpA.

In a coiled coil temperature sensing domain herein described,dimerization involves a dynamic process of forming a dimer of twomonomers involving interactions between residues in positions a to g ofheptad repeats in the temperature sensitive amino acid sequences of eachmonomer protein as will be understood by a skilled person. Inparticular, the hydrophobic residues at positions a and d interact witheach other and form the hydrophobic core or interface of the coiledcoils, and are mainly responsible for the formation and stability of thecoiled-coil. Electrostatic attractions at positions d-e and g-e canprovide additional stability to the system by encouraging the saltbridge formation between the two coils.

The term “electrostatic interactions” as used herein, refers tointeractions between static electrically charged particles, an inparticular between amino acids, wherein amino acids can be coulombicallyattracting or coulombically repelling, according to Coulomb's law (seeEq. 4). Electrostatic interactions comprise ionic interactions, hydrogenbonding, halogen bonding, Van der Waals forces, dipole-dipole,dipole-induced dipole, hydrophobic effects, and others as understood bya skilled person. An amino acid replacement that changes the coulombicforce (in pN) between two amino acids residues by changing from positiveto negative or vice-versa, or increases the coulombic force in eitherdirection, can change the structure of a polypeptide or protein, orprotein-protein interactions, and related functional characteristics ofthe polypeptide or protein.

Formation of these interchain salt bridges can also shield the non-polarcore from solvent, further stabilizing the coiled-coils. Positions e andg of the heptad repeat flank the hydrophobic interface of thecoiled-coil and can contribute to the hydrophobicity of the core byfolding over the interface to interact with the hydrophobic residuesthrough their side chain methylene groups, thereby shielding the corefrom water. Examples of proteins including coiled coil temperaturedomain include transcription factors such as lac repressor, TlpAprotein, KfrA and others identifiable by a skilled person and otherproteins not involved in regulation of gene expression, such as myosin,tropomyosin, and others as will be identified by a skilled person.

A representative example of coiled coil temperature sensitivetranscription factors, is TlpA a transcriptional autorepressor from thevirulence plasmid of Salmonella typhimurium. This protein contains anapproximately 300 residue C-terminal coiled-coil domain that undergoessharp, temperature-dependent uncoiling between 37° C. and 45° C., and anN-terminal DNA binding domain that, in its low-temperature dimericstate, blocks transcription from the ˜50 bp TlpA operator/promoter [1,20].

As shown in Example 1, the TlpA operator is a strong promoter (88-foldstronger than LacI^(Q)) driven by the transcription factor σ⁷⁰. Thispromoter has bidirectional activity with identical thermal regulation inboth orientations, but approximately 200-fold lower maximal expressionin the reverse direction [21].

TlpA is expected to be more orthogonal to cellular machinery than boththe native heat shock promoters and the engineered TetR and LacIrepressors, the latter of which are utilized in multiple endogenous andengineered gene circuits [22-24]. A homology search [25] showed thatTlpA and TcI repressors are present in far fewer bacterial species thaneither TetR or LacI (FIGS. 3A-3B).

In absence of a crystal structure of TlpA protein, structural modelingsoftware enables prediction of structural features of TlpA and similarproteins. The primary sequence of TlpA protein is shown in FIG. 11B. Thesecondary structure of the TlpA is predicted using JPred, a proteinsecondary structure prediction software [26], and shown in FIG. 17. Inparticular, FIG. 17 shows the amino acid sequence of TlpA protein, withamino acids annotated with ‘H’ on the lower row representing an aminoacid that forms part of a predicted heptad repeat of a coiled-coileddomain. The secondary structure prediction of TlpA was performed usingthe software JPred. This software predicts that the DNA binding domainof TlpA consists of three alpha helices separated by short linkers.

Structure prediction servers such as COILS [4], Paircoil 2 [5] andLOGICOIL [6] and Jpred among other as will be understood by a skilledperson can be further used for analysis of coiled coil domains.Graphical depictions of coiled coils can be produced using software suchas DrawCoil 1.0 [7]. Examples of structural analysis results of TlpAusing these programs are shown in FIG. 14, FIG. 15, FIGS. 16A-16M, andFIG. 17.

FIGS. 14 and 15 shows the amino acid sequence of TlpA divided intoheptad repeat portions ‘A’ through ‘M’ in the predicted coiled-coildomain, with the coil register of heptads assigned based on consensusbetween previous literature [3] and the structure prediction serversPaircoil 2 [5] and LOGICOIL [6]. In particular, the register positionwithin the heptad repeat for each predicted heptad repeat portion ‘A’through ‘M’ is indicated; for example “A starts at position b” (see FIG.14). In FIG. 14, the amino acid position number within each heptadportion of the coiled coil domain and within the DNA binding domain isindicated above each upper row, while the amino acid position numberwithin the whole TlpA protein sequence is indicated above each lowerrow. The symbol “*” indicates the stop codon at the C-terminus of theTlpA protein. An arrowhead symbol ‘>’ at the end of each line indicatethe direction of the amino acid sequence.

FIGS. 16A-16M shows ‘helical wheel’ diagrams of the predicted structureof the TlpA coiled-coil interface as viewed down the long axis of thehelix, with two TlpA proteins shown side-by-side, in a predicted dimerconfiguration of the transcriptionally repressive state. Each helicalwheel diagram (FIGS. 16A-16M show a portion of the predicted coiled-coildomain, divided into uninterrupted amino acid sequences that arepredicted to fit into the same heptad register, according to thesequences listed in FIG. 15, as follows: FIG. 16A corresponds to FIG. 15Heptad repeat ‘A’; FIG. 16B corresponds to FIG. 15 Heptad repeat ‘B’;FIG. 16C corresponds to FIG. 15 Heptad repeat ‘C’; FIG. 16D correspondsto FIG. 15 Heptad repeat ‘D’; FIG. 16E corresponds to FIG. 15 Heptadrepeat ‘E’; FIG. 16F corresponds to FIG. 15 Heptad repeat ‘F’; FIG. 16Gcorresponds to FIG. 15 Heptad repeat ‘G’; FIG. 16H corresponds to FIG.15 Heptad repeat ‘H’; FIG. 16I corresponds to FIG. 15 Heptad repeat ‘I’;FIG. 16J corresponds to FIG. 15 Heptad repeat; FIG. 16K corresponds toFIG. 15 Heptad repeat ‘K’; FIG. 16L corresponds to FIG. 15 Heptad repeat‘L’; FIG. 16M corresponds to FIG. 15 Heptad repeat ‘M’. The predictedalpha-helical heptad repeat (labeled a-b-c-d-e-f-g) is shown, connectedby progressively thinner straight lines shown in an N-terminal toC-terminal direction. A dashed line is shown between the last residue ofa heptad and the first residue of a next heptad in a portion of theheptad repeat. Single-letter amino acid symbols shown circled at eachposition in a heptad. The sequence of amino acids in an N-terminal toC-terminal direction are shown at each position of a heptad, with thefirst heptad in the portion of the heptad repeat shown on the line ofeach large circle representing an alpha-helix, and the amino acids ofconsecutive heptads are shown further out from the large circle Bluedashed lines represent predicted energetically favorable ionicinteractions; red dashes indicate predicted repulsive ionicinteractions. The coil register was assigned based on the COILS serverin the www.ch.embnet.org/cgi-bin/COILS_form_parser webpage. The imageswere produced using DrawCoil 1.0 [7].

In some embodiments, the temperature sensitive transcription factor is aglobular temperature sensitive factor, and the temperature-sensingdomains contain two globular monomers forming a dimer by interactionsbetween the C-terminal domains (CTDs) of the two monomers. The term“globular protein” indicates spherical, globe-like proteins induced bythe proteins' tertiary structure, comprising a core interface and anexterior solvent-exposed face. The term “interface” as used herein inconnection with globular temperature sensitive transcription factors,and in general with homodimers of the disclosure, indicates a portion ofa monomer protein comprising amino acids involved in the cooperativebinding of the monomer protein with the other monomer protein formingthe homodimer. The term “core interface” as used herein refers to aminoacid residues centrally located within a dimerization domain at theinterface of dimerized proteins The term “exterior solvent exposed face”indicates a portion of the monomer protein comprising amino acid outsidethe interface and interacting with the solvent. Amino acid residuestypically found at the interface are hydrophobic, but can also comprisepolar or charged amino acids. Amino acids comprising the exteriorsolvent exposed face are usually polar or charged, but can behydrophobic. The non-polar (hydrophobic) amino acids are generallybounded towards the molecule's interior whereas polar, hydrophilic aminoacids are generally bound outwards, allowing dipole-dipole interactionwith the solvent, thus contributing to the molecule's solubility.Globular protein structure can be determined by using techniquesincluding ultracentrifugation, dynamic light scattering, and othertechniques known to those skilled in the art.

In globular temperature sensitive transcription factors herein describedthe temperature-sensing domain comprises a homodimeric globularstructure, in which each monomer comprises a C-terminal domain (“CTD”)of about 105 residues in length The CTDs mediates the dimerization ofthe temperature-sensing domain in the thermal transcription bioswitches,which cooperatively leads to the dimerization of the DNA-binding domainconnected to the temperature-sensing domain and subsequently the bindingof the DNA-binding domain to the recognition sequence of the DNA.

In the globular temperature sensitive transcription factor, thetemperature sensitive domain of each monomer protein has a length of 105amino acids. and comprises an A bend, a B bend, a C bend, a D bend, an Ebend, an F bend, a G bend, an H bend, an I bend, a J bend, a K bend, anL bend, an M bend, a βA strand, a βB strand, a βC strand, a βD strand, aβE strand, a βF strand, an A turn, a B turn, a C turn, and a βA bridgelinked one to another by loop regions. The globular temperature sensingdomain can have has a sequence

X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀-X₅₁-X₅₂-X₅₃-X₅₄-X₅₅X₅₆-X₅₇-X₅₈-X₅₉-X₆₀-X₆₁-X₆₂-X₆₃-X₆₄-X₆₅-X₆₆-X₆₇-X₆₈-X₆₉-X₇₀-X₇₁-X₇₂-X₇₃-X₇₄-X₇₅-X₇₆-X₇₇-X₇₈-X₇₉-X₈₀-X₈₁-X₈₂-X₈₃-X₈₄-X₈₅-X₈₆-X₈₇-X₈₈-X₈₉-X₉₀-X₉₁-X₉₂-X₉₃-X₉₄-X₉₅-X₉₆-X₉₇-X₉₈-X₉₉-X₁₀₀-X₁₀₁-X₁₀₂-X₁₀₃-X₁₀₄-X₁₀₅(SEQ ID NO: 457)whereinX₁ can be a polar residue defining an N-terminal residue;X₂ can be a polar residue forming the A bend;X₃ to X₅ can be polar or charged residues forming a loop;X₆ to X₈ can be polar amino acids forming the B bend;X₉ can be polar amino acid forming a loop;X₁₀ to X₁₃ can be any amino acids forming the βA strand;X₁₄ to X₁₅ can be any amino acids forming a loop;X₁₆ to X₁₉ can be polar residues forming the C bend;X₂₀ to X₂₂ can be polar or non-polar amino acids forming a loop;X₂₃ to X₂₄ can be polar amino acid residues forming the D bend;X₂₅ can be a non-polar amino acid forming a loop;X₂₆ to X₂₇ can be polar or ionic amino acids forming the E bend;X₂₈ to X₃₀ can be polar or non-polar amino acids forming a loop;X₃₁ to X₃₂ can be polar, non-polar or ionic amino acids forming the Fbend;X₃₃ can be any amino acid forming a loop;X₃₄ to X₃₇ can be non-polar amino acids forming the βB strand;X₃₈ to X₃₉ can be any amino acids forming a loop;X₄₀ to X₄₁ can be any amino acids forming the G bend;X₄₂ to X₄₄ can be any amino acids forming a loop;X₄₅ to X₄₆ can be any amino acids forming the A turn;X₄₇ can be a non-polar amino acid forming the H bend;X₄₈ to X₅₂ can be polar, non-polar, or ionic amino acids forming the βCstrand;X₅₃ can be any amino acid forming the I bend;X₅₄ to X₅₆ can be any amino acids forming the B turn;X₅₇ to X₆₀ can be any amino acids forming a loop;X₆₁ to X₆₄ can be any amino acids forming the βD strand;X₆₅ to X₆₆ can be any amino acids forming a loop;X₆₇ to X₆₉ can be any amino acids forming the J bend;X₇₀ can be any amino acids forming a loop;X₇₁ to X₇₃ can be any amino acids forming the βE strand;X₇₄ can be any amino acid forming a loop;X₇₅ to X₇₆ can be any amino acids forming the K bend;X₇₇ to X₇₈ can be polar amino acids forming the C turn;X₇₉ can be a polar amino acid forming the L bend;X₈₀ to X₈₁ can be polar amino acids forming a loop;X₈₂ can be a polar amino acid forming the βA bridge;X₈₃ to X₈₈ can be polar amino acids forming a loop;X₈₉ to X₉₆ can be polar or ionic amino acid residues forming the βFstrand;X₉₇ to X₁₀₀ can be polar or non-polar amino acids forming a loop;X₁₀₁ to X₁₀₃ can be polar amino acids forming the M bend;X₁₀₄ to X₁₀₅ can be polar or non-polar amino acids defining a C-terminalsegment.

In particular in which the globular temperature sensing domain is adimer of two monomers, each containing a globular structure having acore interface and exterior solvent exposed face. Each monomer interactswith the corresponding portion of the other monomer through chemicaland/or physical interactions at the core interface to form a globulartemperature sensitive transcription factor. In those embodiments,cooperative unfolding of the monomers results in a loss of the abilityto correctly position the two halves of the DNA binding domain found atthe N-termini of each protein monomer. Tuning of the thermal responsecurve is achieved by modulating the affinity of the two monomers.

In globular sensitive temperature sensing domain the dimerization ofeach monomer to form the domain in the dimer, involves interactions ofsecondary structures within the two monomers driving dimerization. Inparticular, a single-strand alpha-helix or a random coil can interactwith a corresponding alpha-helix in another monomer to form adouble-strand coiled-coil interaction domain. In a globular structurecomprising segments, loops, bends, β-strands, turns, and β-bridges, eachstructure can interact with the corresponding portion of the othermonomer through chemical and/or physical interactions at the coreinterface to form a dimer as will be understood by a skilled person.

In a globular sensitive temperature sensing domain of SEQ ID NO:457residues X₁, X₂, X₂₀ to X₂₂, X₂₅, X₂₈ to X₃₀, X₃₁ to X₃₂, X₃₄ to X₃₇,X₄₇, X₄₈ to X₅₂, X₇₇ to X₇₈, X₇₉, X₈₀ to X₈₁, X₈₂, X₈₃ to X₈₈, X₈₉ toX₉₆, X₉₇ to X₁₀₀, and X₁₀₄ to X₁₀₅ are located in the globulartemperature sensitive domain interface between the two monomer proteins:

In a globular sensitive temperature sensing domain of SEQ ID NO:457residues X₃ to X₅, X₆ to X₈, X₉, X₁₆ to X₁₇, X₂₃ to X₂₄, X₄₂ to X₄₄, X₄₅to X₄₆, X₅₃, X₅₄ to X₅₆, X₅₇ to X₆₀, X₆₇ to X₆₉, X₇₀ are solvent exposedamino acids:

In a globular sensitive temperature sensing domain of SEQ ID NO:457residues X₁₀₁ to X₁₀₃ in SEQ ID NO:457 are polar residues coordinated toother polar residues by hydrogen bonding, and have structuralcharacteristics similar to polar residues at the interface between thetwo monomers

In some embodiments, the globular temperature sensing domain can alsoinclude a variant of SEQ ID NO: 457 in which any of the amino acidresidues of SEQ ID NO: 457 is substituted with a ΔΔG of substitution ofa Rosetta modeling software greater than −0.5 Rosetta Energy Unit (REU)or lower than 0.5 Rosetta Energy Unit (R.E.U).

The term “Rosetta Energy Unit” or “R.E.U.” as used herein indicates aunit to measure rosetta energy on an arbitrary scale using the Rosettasoftware package.

In particular, Rosetta software package includes algorithms forcomputational modeling and analysis of macromolecular structures as wellas tools for structure prediction, design and remodeling of proteins andnucleic acids (http://www.rosettacommons.org). In particular, RosettaBackrub model (see the website http://kortemmelab.ucsf.edu/backrub atthe filing date of the present disclosure) implements the Backrubmethod, derived from observations of alternative conformations inhigh-resolution protein crystal structures, for flexible backboneprotein modeling. Backrub modeling is applied to three relatedapplications using the Rosetta program for structure prediction anddesign: (i) modeling of structures of point mutations, (ii) generatingprotein conformational ensembles and designing sequences consistent withthese conformations and (iii) predicting tolerated sequences atprotein-protein interfaces. Detailed description about the Rosettasoftware package and RosettaBackrub can be found in related literaturesuch as Lauck F. et al. (2010) [27]. As a person of ordinary skill inthe art would understand, the program can be used to automaticallygenerate libraries of sequence variations for protein interfaces thatcan be further screened experimentally for changes in proteininteraction affinity.

In particular, RosettaBackrub can create ensembles of structures forflexible backbone modeling. This method has been derived fromobservations of alternative conformations in high-resolution crystalstructures and involves local backbone rotations about axes between Caatoms of protein segments. In general, first, a segment of typically2-12 residues is randomly selected. Then, all atoms of this fragment arerotated as a rigid body by an angle of up to 11°-40° around the axisbetween the two C_(α) pivot atoms (see the web server onlinedocumentation section for details). Backrub moves, interleaved withrearrangements of the surrounding side chains, are sampled by a MonteCarlo algorithm using the Rosetta all-atom force field. Backrub and sidechains moves are made 10000 times, with each new conformation beingscored with the Rosetta scoring function. The resulting score is used todetermine whether the new conformation is accepted or not according tothe Metropolis criterion. In order to create an ensemble of size N thisalgorithm is independently applied N times to the input structure.Side-chain sampling and scoring in sequence design are as described inKuhlman et al. (2003) [28]

The Protein Interface Sequence Plasticity Prediction model ofRosettaBackrub predicts the sequence diversity (also referred to as“plasticity”) in protein-protein interfaces. This application models thetolerated sequence space for interface positions in a protein complex[29] such as the interface of SEQ ID NO: 459. First, Backrub is appliedto the two interaction partners in a protein-protein complex to create aconformational ensemble. Backbone flexibility is modeled at allpositions in each complex partner. Each of the resulting complexstructures is then subject to the sequence plasticity protocol. Thisprotocol uses a genetic algorithm to sample amino acid changes atinterface positions specified by the user. Modeled residues are scoredaccording to their contributions to the stability of the proteinpartners as well as to the stability of the protein-protein interface.Interface sequences are recorded and kept if their score is within athreshold from the interface and complex scores of the sequence in theinput file, as described in Humphris et. al. The input for theapplication model is a single PDB structure and the output is the PDBfiles of the generated ensemble, as well as the sequences andfrequencies of amino acids of the designed ensemble.

The rosetta energy function used in the Rosetta software package is alinear sum of individually weighted terms shown below. Detaileddescription of the rosetta energy function can be found in relatedliteratures such as Renfrew et. al. (2012) [30]. In brief, the rosettaenergy function contains a physically-based inter-residue Lennard-Jonesterm split into repulsive and attractive components (E_(inter_rep) andE_(inter_atr)), a implicit solvation term implemented as described byLazarids and Karplus (E_(solvation)), knowledge-based reside pairelectrostatics term (E_(pair)), orientation dependent hydrogen bondingterm (E_(sc/bb hb), E_(bb/bb hb) and E_(sc/sc hb)), a knowledge-basedterm that measures the internal energy of an amino acid based onprobabilities from rotamer libraries (the rotamer internal energy term,E_(dunbrack)), a knowledge-based term that measures Ramachandranbackbone torsion preferences of a position (the rama term, E_(rama)),and a reference energy term that represents the energy of the unfoldedstate of a protein (E_(ref)).

E_(protein) = W_(inter  rep)E_(inter  rep)W_(interate)E_(interate) + W_(solvation)E_(solvation) + W_(bb/sc  hb)E_(bb/sc  hb) + W_(bb/bb  hb)E_(bb/bb  hb) + W_(ac/sc  hb)E_(ac/sc  hb) + W_(pair)E_(pair) + W_(dunbrack)E_(dunbrack) + W_(rama)E_(rama) + W_(reference)E_(reference)

The rosetta energy obtained from the above equation is on an arbitraryscale, also referred to as Rosetta Energy Unit (“REU”). In general,structures with lower rosetta energies are considered to be more stablethan structures with higher energies. The rosetta energy differencebetween two structures can be defined as ΔΔG, which is defined asE_(protein2)−E_(protein1). Protein1 can be a WT while protein2 can be avariant of the WT with a single or multiple amino acid substitutions.

Therefore, the term ΔΔG as used herein indicates the rosetta energydifference between two structures defined as E_(protein2)−E_(protein1).In variants herein described Protein1 can be a WT while protein2 can bea variant of the WT with a single or multiple amino acid substitutions.

In some embodiment, the globular temperature sensing domain, eachmonomer protein can have a globular temperature sensing amino acidsequenceTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVGKVIASQWPEETFG (SEQ ID NO: 459), or a variantof SEQ ID NO: 457 in which any of the amino acid residues of SEQ ID NO:459 is substituted with a ΔΔG of substitution greater than −0.5 RosettaEnergy Unit (REU) or lower than 0.5 Rosetta Energy Unit (R.E.U).

A representative example of a globular sensing domain is atemperature-sensitive variant of the bacteriophage λ repressor cI(mutant cI⁸⁵⁷ containing an A67T mutations, herein referred to as TcI)acting on a tandem pR-pL operator-promoter. TcI repression has beenmodulated via large changes in temperature (e.g., steps from 30° C. to42° C.), rather than a sharper switching. The cI repressor ofbacteriophage λ is another example of a protein that binds to itsoperator sites cooperatively. The C-terminal domain of the repressormediates dimerization as well as a dimer-dimer interaction that resultsin the cooperative binding of two repressor dimers to adjacent operatorsites. Detailed structural information is available for the isolateddomains of the cI repressor [31] and intact dimeric cI repressor boundto an operator sequence [8]

The TcI protein is composed of two structurally distinct domains thatare tethered by a protease sensitive connector. The N-terminal DNAbinding domain (residues 1-92 of FIG. 21) which contains ahelix-turn-helix DNA-binding motif, is a compact alpha-helical domainthat weakly self-associates to form a dimer. Dimers of the DNA bindingdomain recognize and bind to the operator sequences using thishelix-turn-helix motif. The C-terminal domain (residues 132-236; FIG.21), otherwise referred to as the “globular dimerization domain” or“globular domain” is a highly twisted beta-sheet structure that isresponsible for establishing the essential dimer contacts and formediating the higher-order dimer-dimer interactions that underliecooperative binding to the DNA. In addition, the C-terminal domainperforms a self-cleavage reaction, which is triggered in bacteriophagelambda when the lysogenic cell suffers DNA damage and depends upon anactivated form of the bacterial RecA protein. This self-cleavagereaction inactivates the repressor by separating the N-terminal domainfrom the C-terminal domain. The connector (residues 93-131; FIG. 21)which contains the cleavage site, consists of a small protease sensitivelinker and the cleavage site region. Structurally the cleavage siteregion is an integral part of the C-terminal domain, forming a pair ofantiparallel beta-strands that drapes across its surface. Cleavageoccurs at a specific site (between Ala111 and Gly112) within a long loop(residues 106-126) that connects the antiparallel beta strands of thecleavage site region. A depiction of the crystal structure of TcI isshown in FIGS. 22A-22D. The crystal structure of the wild type lambdarepressor (Protein Data Bank (PDB) code 3BDN) was used as the homologymodel for TcI [8].

In transcriptional bioswitch dimers herein described, two monomerproteins configured to bind to one another to form a dimer in a targetenvironment comprising a DNA polynucleotide having a DNA coding regionunder control of a DNA regulatory region, the dimer configured to have aDNA-bound state and an DNA-unbound state with respect to specificbinding of the dimer to the DNA polynucleotide in the targetenvironment.

The temperature sensing domain of the transcriptional bioswitch dimercontrols dimer formation as well as binding and unbinding of thetranscription factor from DNA and the related conversion from a DNAbound state to a DNA-unbound state.

In particular, in each temperature sensing domain of transcriptionalbioswitch dimers herein described, each monomer proteins are configuredto bind to one another with a target environment with a binding constantKd≤100 nM in the DNA-bound state and ≥10 uM in the DNA-unbound statewherein

$\begin{matrix}{K_{d} = e^{(\frac{\Delta\; G}{RT})}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

in which R is the gas constant, T is the temperature of the targetenvironment and ΔG is the molar Gibbs free energy.

In each temperature sensing domain of transcriptional bioswitch dimersherein described, each monomer protein is further configured to bind toone another in the target environment with a thermal Hill coefficientabove 15 to form the dimer in a temperature dependent manner.

In some embodiments, the two monomer proteins of the temperature sensingdomain are further configured to bind to one another in the targetenvironment with a thermal Hill coefficient from 15 to 40 to form thedimer in a temperature dependent manner.

In particular in coiled coil and globular transcriptional bioswitchdimers herein described, the dimerization or de-dimerization of twomonomers exhibits a cooperative behavior, also referred to as“cooperativity”. Cooperativity occurs in molecular structures containingmultiple binding sites. In general, cooperativity describes the changesin conformation or binding energy that occur when a binding site of oneof these structures is activated or deactivated effecting the otherbinding sites in the same molecule. It can also be described as theincreasing (positive cooperativity) or decreasing (negativecooperativity) affinity for binding of the other sites affected by theoriginal binding site. Cooperativity can occur in enzymes, receptors,DNA and many molecules that are made of identical or near identicalsubunits. An example of positive cooperativity can be seen on thebinding of oxygen to hemoglobin to form oxyhemoglobin. Another exampleis the unwinding of DNA in which sections of DNA first unwind followedby the process of unwinding another group of adjacent nucleotides.Similar processes also apply to other types of chain molecules, such asthe folding and unfolding of alpha-helices in coiled-coils of thetemperature-sensing domain.

In embodiments herein described, the cooperativity of atemperature-sensing domain can be quantified by a single parameterreferred to as “Hill coefficient”. The Hill coefficient is a measure forthe cooperative of the temperature-sensing domain de-dimerizationtransition. High Hill coefficients go together with sharpde-dimerization transitions while low Hill coefficients indicate agradual transition from the folded dimer to unfolded two monomerconformation. The Hill coefficient can be mathematically calculated fromfitting a circular dichroism (CD) melting curves as follow:

$\begin{matrix}{{f(T)} = \frac{{aT}^{b}}{T_{m}^{b} + T^{b}}} & {{Eq}.\mspace{14mu} 2}\end{matrix}$

where T_(m) is the melting temperature of a temperature-sensing domain,a the amplitude, T the temperature and b the Hill coefficient.

The melting temperature “Tm” of a temperature sensing binding domain isthe temperature, at which the temperature sensing binding domain withina temperature sensitive transcription herein described denaturates. Thechange in size or structure that accompanies the protein denaturationcan be identified using DLS techniques, CD techniques and othertechniques identifiable by a skilled person. Factors affecting the Tm ofa temperature sensing domain comprise the primary sequence of aminoacids and environment conditions, e.g. pH and salt concentration, aswell as post translational modifications, e.g. glycosylation, andformation of complex with other molecules (proteins or DNA) or otherfactors that can affect the stability of the protein structure and hencethe melting temperature as will be understood by a skilled person.

As a person skilled in the art would understand, CD and otherspectroscopic measurements that measure changes in absorption andfluorescence collected as a function of temperature can determine thethermodynamics of protein unfolding and binding interaction. Forexample, measuring CD as a function of temperature can be used todetermine the effects of mutations on protein stability, as well as thebinding constants of interacting proteins and protein-ligand complexes.

In some embodiments, a CD melting curve can be recorded usingspectroscopic technique for following the de-dimerization anddimerization of temperature-sensing domains as a function oftemperature, as will be understood by a person skilled in the art (seeExample 6).

In coiled coil temperature sensitive transcription factors hereindescribed, the temperature-sensing domains in the sense of the currentdisclosure contain a two-stranded α-helical coiled-coil structure thathave sharp uncoiling transitions with a Hill coefficient above 15. Insome embodiments, the two monomer proteins of the coiled coiltemperature sensing domain are further configured to bind to one anotherin the target environment with a thermal Hill coefficient from 15 to 40to form the dimer in a temperature dependent manner. For example, thecoiled-coil domain of TlpA has a Hill coefficient of from about 15 to 25(see Example 6).

For example a sharp transition of TlpA protein dimerization betweencoiled coil domains of each monomer features cooperative binding, asindicated by the Hill coefficient (see Example 6). Identification ofproteins having similar cooperative dimer binding can be done usingtechniques including circular dichroism (CD) spectroscopy andcalculating Hill coefficient from fitting a circular dichroism (CD)melting curve, as previously described.

In globular temperature sensitive transcription factors hereindescribed, the two globular monomers forming a dimer by interactionsbetween the C-terminal domains (CTDs) of the two monomers, also havesharp de-dimerization transitions with a Hill coefficient above 15. Insome embodiments, the two monomer proteins of the globular temperaturesensing domain are further configured to bind to one another in thetarget environment with a thermal Hill coefficient from 15 to 40 to formthe dimer in a temperature dependent manner.

In temperature sensitive transcription factors herein described and inparticular in coiled coil and globular temperature sensing domain, theTm of the temperature sensing domain also controls the temperature ofthe target environment at which the temperature sensitive transcriptionfactor is converted from the DNA bound state to the DNA unbound stateherein also bioswitch temperature or Tbs), determined by the meltingtemperature Tm of the temperature sensitive binding domain.

In particular in temperature sensitive transcription factors in whichthe temperature sensing domain is a coiled-coil interaction domainessentially consisting of supercoiled alpha-helical structure,cooperative unfolding of the coil results in a loss of the ability tocorrectly position the two halves of the DNA binding domain found at theN-termini of each protein chain.

In temperature sensitive transcription factors in which the temperaturesensing domain is a globular interaction domain each monomer interactswith the corresponding portion of the other monomer through chemicaland/or physical interactions at the core interface to form a globulartemperature sensitive transcription factor. In those embodiments,cooperative unfolding of the monomers results in a loss of the abilityto correctly position the two halves of the DNA binding domain found atthe N-termini of each protein monomer.

In particular, temperature sensitive transcription factors hereindescribed and more particularly in coiled coil and globular temperaturesensing domain, the Tm of the temperature sensing domain defines thebioswitch temperature of the temperature sensitive transcription factor(Tbs) herein also indicated as threshold temperature, the Tbs being atemperature of the target environment at which the temperature sensitivetranscription factor is converted from the DNA bound state to the DNAunbound state, with Tbs=Tm+0° C. to 5° C. In particular, Tbs=Tm+0° C. to5° C. in a target environment with a net concentration of monomerproteins from 2 to 20 uM.

In some embodiments, the melting temperature Tm of the temperaturesensing domain of a coiled coil or globular temperature sensing domainherein described can be Tm=from 20 to 80° C. In some embodiments, themelting temperature Tm of the temperature sensing domain of a coiledcoil or globular temperature sensing domain herein described can beTm=from 25 to 60° C. In some embodiments the melting temperature Tm ofthe temperature sensing domain of a coiled coil or globular temperaturesensing domain herein described can be Tm=from 30 to 50° C. In someembodiments, the melting temperature Tm of the temperature sensingdomain of a coiled coil or globular temperature sensing domain hereindescribed can be Tm=from 32 to 46° C.

In some embodiments, the Tbs can be Tbs=Tm+2.5 to 5° C., wherein thetemperature sensitive transcription factor is encoded in the targetenvironment by a polynucleotide in a number from 100 to 1000 copies percell. In some embodiments, the Tbs can be Tbs=Tm+1 to 3.5° C., whereinthe temperature sensitive transcription factor is encoded in the targetenvironment by a polynucleotide in a number from 10 to 100 copies percell. In some embodiments, the Tbs can be Tbs=Tm+0 to 1.5° C., whereinthe temperature sensitive transcription factor is encoded in the targetenvironment by a polynucleotide in a number below 10 copies per cell.

The bioswitch temperature of temperature sensitive transcription factorsaffects the related bioswitch properties in a target environment whereinthe temperature sensitive transcription factor's conversion from aDNA-bound state to n DNA-unbound state with respect to specific bindingof the dimer to a DNA polynucleotide can be used to activate orinactivate expression of target genes possibly included in one or moregenetic circuits.

In some embodiments, the bioswitch temperature of the coiled coil andglobular transcriptional bioswitch dimers herein described can beincreased or decreased through modification of the amino acid sequencesof the temperature sensing domain to obtain temperature sensitivetranscription factors that can operate at controlled temperatures.

In particular, thermal bioswitches operating at controlled temperaturecan be created by modifying the temperature sensing domain to result inmodulation of the temperature response profile to higher or lowertemperatures, as well as in changing the profile from a cooperative,switch-like induction to a linear “analog” transition. In some of thoseembodiments, a temperature sensitive transcription factor can bemodified thermal transcriptional bioswitches can be mutated and “tuned”in the sense of the disclosure, to increase or decrease their bioswitchtemperature Tbs and activate at different transition temperatures. Inparticular, in some embodiments, the thermal transcriptional bioswitchescan be tuned to activate at new temperatures while retaining sharp,robust switching performance.

In particular, temperature sensing domain of the transcriptionalbioswitch dimers obtainable with methods herein described can beconfigured so that the thermal bioswitches can be tuned to exhibit an ONor OFF state at a particular temperature range while still retaining asharp thermal transition resulting in a large change in activity. Forexample, modification of the temperature sensing domain of a startingcoiled coil or globular transcriptional bioswitch dimer can be performedto obtain a >100-fold difference between an on and off state, and a10-fold switching over a temperature range less than 5° C. Amodification of the temperature sensing domain of a coiled coil ortranscriptional bioswitch dimers herein described can be performed toobtain a transcription factor with a Tbs bioswitch temperature thatselected for specific application such as tunable thresholds within abiomedically relevant range of 32° C. to 46° C. Accordingly, one or moretemperature sensitive transcription factor can be provided starting fromcoiled coil or globular transcriptional bioswitch dimers for use withina cell can be provided that are orthogonal to endogenous cellularmachinery and compatible with other thermos-responsive components and aTbs compatible with the cell physiological temperature to allowmultiplexed thermal logic.

In some embodiments, tuning of the thermal response curve is achieved bymodulating the affinity of the two coiled coil strands for each other.In those embodiments modification of a bioswitch temperature Tbs of acoiled coil temperature sensitive transcription factor can be performedby providing a coiled coil transcriptional bioswitch dimer hereindescribed having a starting bioswitch temperature Tbs₀ in the targetenvironment and two monomer proteins configured to form a temperaturesensing domain in the target environment with a starting meltingtemperature Tm₀; and replacing in at least one monomer protein of thetwo monomer proteins forming the temperature sensing domain one or moreresidues in positions a, b, d, e and g of a heptad repeat in thetemperature sensing amino acid sequence of the provided coiled coiltemperature sensitive transcription factor.

In particular in some embodiments the replacing can be performed byreplacing at least one of a hydrophobic amino acid in a position aand/or d of an heptad repeat of the temperature sensitive amino acidsequence of the temperature sensing domain with residues configured toincrease or decrease hydrophobic packing between corresponding aminoacid residues in positions a and/or d of the two monomer formingtemperature sensing domain.

The term “hydrophobic packing” as used herein relates to the aggregatingtogether of nonpolar molecules, and in particular, amino acids, toreduce the surface area exposed to water and minimize their disruptiveeffect. The efficiency of hydrophobic packing can be quantified bymeasuring the partition coefficients of non-polar molecules betweenwater and non-polar solvents. The partition coefficients can betransformed to free energy of transfer which includes enthalpic andentropic components:

ΔG=ΔH−TΔS  Eq. 3

where G is the Gibbs free energy of protein folding, H is the totalenthalpy of a system, T is temperature, and S is entropy. Thesecomponents can be experimentally determined using techniques such ascalorimetry, circular dichroism or NMR, where an increase in ΔGindicates a decrease in efficiency of hydrophobic packing

In some embodiments the replacing can be performed by replacing at leastone polar or charged amino acid in position b e, and/or g of at leastone heptad repeat of the temperature sensing domain amino acid sequence,with a hydrophobic residue,

In some embodiments the replacing can be performed by replacing at leastone polar or charged amino acid in positions e and/or g of at least oneheptad repeat of the temperature sensing domain amino acid sequence witha residue configured to increase or decrease coulombic repulsion betweencorresponding residues in positions a, d, e and/or g of at least oneheptad repeat of the temperature sensing domain amino acid sequence ofthe two monomers forming the temperature sensing domain.

In some embodiments the replacing can be performed by replacing one ormore amino acid residues in anyone of positions a, b, d, e and/or g ofat least one heptad repeat of the temperature sensing domain amino acidsequence as indicated above in combination one with the other.

In embodiments of the method to modify the amino acid sequences of thetemperature sensing domain of coiled coil transcriptional bioswitchdimers herein described, the replacing is performed to obtain a variantof the coiled coil temperature sensitive transcription factor with amelting temperature of the temperature sensing domain Tm_(m) lower orhigher than Tm₀ in the target environment, the obtained variant having abioswitch temperature Tbs_(m) lower or higher than Tbs₀ in the targetenvironment.

In particular, some of those embodiments coiled coil temperaturesensitive transcription factors of the disclosure can be engineered tolower the bioswitch temperature Tbs of a starting coiled coiltranscriptional bioswitch dimer, by replacing

a polar amino acid in a position b of at least one heptad repeat of thetemperature sensing domain amino acid sequence with a hydrophobic aminoacid,

a hydrophobic amino acid in a position d at least one heptad repeat ofthe temperature sensing domain amino acid sequence with a polar aminoacid,

a charged amino acid in a position e at least one heptad repeat of thetemperature sensing domain amino acid sequence with a charged amino acidhaving a pKa different from the original by equal or higher than 0.5 or

at least one of a hydrophobic amino acid in a position a, a hydrophobicamino acid in a position d, a charged amino acid in a position e and acharged amino acid in a position g in at least one heptad repeat of thetemperature sensing domain amino acid sequence with amino acid residuessuch that pairs formed by corresponding residues in positions a, d, e,and g on the two monomer protein interact with a coulombic force F≥1 pN,wherein

$\begin{matrix}{F = {k_{e}\frac{q_{1}q_{2}}{r^{2}}}} & {{Eq}.\mspace{14mu}(4)}\end{matrix}$

where k_(e) is Coulomb's constant (k_(e)=8.99×10⁹ N m² C⁻²), q₁ and q₂are the signed magnitudes of the charges on each amino acid residue ofthe pair of residues, and the scalar r is the distance between thecharges

Exemplary variants of coiled coil transcriptional bioswitch dimersherein described having a lower bioswitch temperature with respect to astarting coiled coil temperature sensitive transcription factor, andobtainable methods herein described comprise dimers comprising a coiledcoil temperature sensing domain of sequence SEQ ID NO: 1 to SEQ ID NO:456 possibly with a 0% to 20% variation and including at least one ofthe replacement herein described in one or more of the amino acidresidues in positions a, b, d e and g of at least one heptad repeat inthe sequence that lower the bioswitch temperature Tbs of the coiled coiltranscriptional bioswitch dimers.

Specific variants lowering threshold temperature of WT TlpA which has athreshold transcriptional activation temperature Tbs above 43° C. can beprovided starting from a WT TlpA which has a threshold transcriptionalactivation temperature Tbs above 43° C. and mutating P60L, D135V(replacing a polar amino acid b in the TlpA WT sequence with ahydrophobic amino acid), K187R K187R (replacing the charged amino acid ein the TlpA WT sequence with an amino acid residue such that pairsformed by residue e on one of the two TlpA monomer units with residue gon another monomer unit interact with a stronger coulombic force),K202I, L208Q (replacing core hydrophobic residue d in the TlpA WTsequence with a polar residue to decrease hydrophobic packing) in TlpA(“TlpA₃₆”) to provide a bioswitch with a threshold transcriptionalactivation temperature centered at 36° C. Mutating D135V (replacing apolar amino acid in position b in the TlpA WT sequence with ahydrophobic amino acid), A217V, L236F (replacing core hydrophobicresidue d in the TlpA WT sequence with another hydrophobic residue withless efficient packing) (“TlpA₃₉”), generates another bioswitch with athreshold transcriptional activation temperature centered at 39° C. (seeExamples 1-2).

In some embodiments coiled coil transcriptional bioswitch dimers hereindescribed can be engineered to increase the bioswitch temperature Tbs ofa starting coiled coil temperature sensitive transcriptional bioswitch,by replacing

at least one of a hydrophobic amino acid in a position a and thehydrophobic amino acid in a position d of at least one heptad repeat ofthe temperature sensing amino acid sequence, with a polar amino acid ora charged amino acid, and/or

at least one of a hydrophobic amino acid in a position a, a hydrophobicamino acid in a position d, a charged amino acid in a position e and acharged amino acid in a position g of at least one heptad repeat of thetemperature sensing amino acid sequence with amino acid residues suchthat pairs formed by corresponding residues in positions a, d, e, and gon the two monomers interact with a coulombic force F≤1 pN calculatedaccording to Equation 3.

Exemplary variants of coiled coil transcriptional bioswitch dimersherein described having a higher bioswitch temperature with respect to astarting coiled coil transcriptional bioswitch dimer, and obtainablemethods herein described comprise dimers comprising a coiled coiltemperature sensing domain of sequence SEQ ID NO: 1 to SEQ ID NO: 456possibly with a 0% to 20% variation and including at least one of thereplacement herein described in one or more of the amino acid residuesin positions a, b, d e and g of at least one heptad repeat in thesequence that increases the bioswitch temperature Tbs of the coiled coiltranscriptional bioswitch dimers.

In some embodiments in which the temperature sensing domain is a dimerof two monomers, each containing a globular structure having a coreinterface and exterior solvent exposed face, tuning of the thermalresponse curve is achieved by modulating the affinity of the twomonomers.

In particular, amino acid residues at the interface the globulartemperature sensing domain and/or solvent exposed residues of theglobular temperature sensing domain can be replaced with polar or moreor less polar residues, charged or more or less charged residues,hydrophobic or more or less hydrophobic residues, or bulky or more orless bulky residues which are identifiable a skilled person upon readingof the present disclosure.

In particular some embodiments, shifting the threshold temperature Tbsof globular temperature sensitive bio switch dimer to lower temperaturesis accomplished by replacing solvent exposed residues in SEQ ID NO:457with more non-polar residues to destabilize the protein. Polar residuesexposed to the solvent that can be replaced with more non-polar residuescomprise the following: X3 to X5, X16 to X17, X23 to X24, X6 to X8, andX9. Residues exposed to the solvent that can be any amino acid and canbe replaced with more nonpolar residues comprise: X33, X42 to X44, X45to X46, X53, X54 to X56, X57 to X60, X67 to X69, and X70.

In some embodiments, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to lower temperatures isaccomplished by replacing nonpolar residues at the interface between thetwo monomers in SEQ ID No:457 with more polar residues to destabilizethe protein. In some embodiments, shifting the threshold to lowertemperatures is accomplished by replacing nonpolar residues at theinterface between the two monomers in SEQ ID NO:457 with larger residuesresulting in less efficient packing and destabilization of the protein.Nonpolar residues at the interface between the two monomers that can bereplaced with more polar residues or with larger residues comprise thefollowing: X25, X34 to X37, X47. Polar, nonpolar or ionic residues atthe interface between the two monomers that can be replaced with morepolar residues or with larger residues comprise the following: X31 toX32, X48 to X52. Polar or ionic residues at the interface between twomonomers that can be replaced with more polar residues or with largerresidues comprise the following: X89 to X96. Polar or non-polar residuesat the interface between two monomers that can be replaced with morepolar residues or with larger residues comprise the following: X20 toX22, X28 to X30, X97 to X100, X104 to X105.

In some embodiments, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to lower temperatures isaccomplished by replacing polar residues at the interface between thetwo monomers in SEQ ID NO:457 with more polar residues to destabilizethe protein. In some embodiments, shifting the threshold to lowertemperatures is accomplished by replacing polar residues at theinterface between the two monomers in SEQ ID NO:457 with larger residuesresulting in less efficient packing and destabilization of the protein.Polar residues at the interface between the two monomers that can bereplaced with more polar residues or with larger residues comprise thefollowing: X1, X2, X77 to X78, X79, X80 to X81, X82, X83 to X88.

In some embodiments, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to lower temperatures isaccomplished by replacing ionic residues at the interface between thetwo monomers in SEQ ID NO:457 with nonionic residues to destabilize theprotein. Polar, non-polar, or ionic residues at the interface betweenthe two monomers that can be replaced with non-ionic residues comprisethe following: X31 to X32, X48 to X52. Polar or ionic residues at theinterface between the two monomers that can be replaced with non-ionicresidues comprise the following: X89 to X96.

In some embodiments, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to lower temperatures isaccomplished by replacing ionic residues near the interface between thetwo monomers of SEQ ID NO: 457 which do not apparently form polar/ioniccontacts with the other monomer with less polar residues to destabilizethe protein. Polar or ionic residues participating in interactions atthe interface between the two monomers that can be replaced with lesspolar residues comprise the following: X26 to X27.

In some of these embodiments, shifting the threshold temperature Tbs ofglobular temperature sensitive bioswitch dimer to lower temperatures isaccomplished by replacing polar residues at the interface in SEQ IDNO:457 with different (e.g. bulkier) polar residues that are stericallyinhibited from forming polar contacts as efficiently as in the originalprotein. Polar, non-polar, or ionic residues at the interface betweenthe two monomers that can be replaced with different (e.g. bulkier)polar residues comprise the following: X31 to X32, X48 to X52. Polar orionic residues at the interface between the two monomers that can bereplaced with different (e.g. bulkier) polar residues comprise thefollowing: X89 to X96.

The term “bulkiness” as used herein, in particular in relation to aminoacids, refers to the molecular weight of an amino acid, wherein aminoacids of higher molecular weight are more bulky. The molecular weight ofthe twenty common amino acids is shown in FIG. 24. Amino acids of MW of140 g/mol can be considered “bulky”, whereas amino acids of MW below 140g/mol can be considered not to be bulky. Thus, for example, replacementof a hydrophobic amino acid with a more bulky hydrophobic amino acid ina polypeptide or protein can change the structure of a polypeptide orprotein, or protein-protein interactions, and related functionalcharacteristics of the polypeptide or protein, such as efficiency ofhydrophobic packing, or others as understood by a skilled person.

In some embodiments, a Polar or ionic residues at residue in theN-terminal DNA binding domain can be replaced with a different (e.g.bulkier). For example, in TcI, residue K68 can be mutated to R to shiftthe threshold from 0 C to 3 C (FIG. 7I).

In some of embodiments, shifting the threshold temperature Tbs ofglobular temperature sensitive bioswitch dimer to lower temperatures isaccomplished by replacing nonpolar residues at the interface between thetwo monomers in SEQ ID NO:457 with bulky sterically occluded, polar, orionic residues to disrupt the interaction. Polar or non-polar residuesat the interface between the two monomers that can be replaced withbulky sterically occluded, polar, or ionic residues comprise thefollowing: X20 to X22, X28 to X30, X97 to X100, X104 to X105. Nonpolarresidues at the interface between the two monomers that can be replacedwith bulky sterically occluded, polar, or ionic residues comprise thefollowing: X25, X34 to X37, and X47.

In some of embodiments, shifting the threshold temperature Tbs ofglobular temperature sensitive bioswitch dimer to lower temperatures isaccomplished by destabilizing the state of the folded structure byreplacing solvent-exposed polar or ionic residues in SEQ ID NO:457 withhydrophobic residues. Solvent-exposed polar residues that can bereplaced with hydrophobic residues comprise the following: X3 to X5, X16to X17, X23 to X24, X6 to X8, and X9. Solvent exposed residues that canbe any amino acid and can be replaced with hydrophobic residues comprisethe following: X33, X42 to X44, X45 to X46, X53, X54 to X56, X57 to X60,X67 to X69, and X70.

In some embodiments, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to lower temperatures isaccomplished by destabilizing the state of the folded structure byreplacing polar residues X101 to X103 of SEQ ID NO: 457 that arecoordinated with each other via hydrogen bonding with nonpolar residuesin the globular temperature sensing domain.

In some embodiments, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to higher temperatures isaccomplished by replacing solvent exposed residues in SEQ ID NO: 457with more polar residues to stabilize the protein. Solvent-exposed polarresidues that can be replaced with more polar residues comprise thefollowing: X3 to X5, X16 to X17 X23 to X24, X6 to X8, X9. Solventexposed residues that can be any amino acid and can be replaced withmore polar residues comprise the following: X33, X42 to X44, X45 to X46,X53, X54 to X56, X57 to X60, X67 to X69, and X70.

In some embodiments, shifting the temperature Tbs of globulartemperature sensitive bioswitch dimer to higher temperatures isaccomplished by replacing nonpolar residues at the interface between thetwo monomers in SEQ ID NO:457 with more non-polar residues to stabilizethe protein. Nonpolar residues at the interface between the two monomersthat can be replaced with more nonpolar residues comprise the following:X25, X34 to X37, X47. Polar, nonpolar or ionic residues at the interfacebetween the two monomers that can be replaced with more nonpolarresidues comprise the following: X31 to X32, X48 to X52. Polar or ionicresidues at the interface between two globular monomers that can bereplaced with more nonpolar residues comprise the following: X89 to X96.Polar or non-polar residues at the interface between two monomers thatcan be replaced with more nonpolar residues comprise the following: X20to X22, X28 to X30, X97 to X100, X104 to X105.

In some embodiments, shifting the threshold to higher temperatures isaccomplished by replacing polar residues at the interface between thetwo monomers in SEQ ID NO:457 with more non-polar to stabilize theprotein. Polar residues at the interface between the two monomers thatcan be replaced with more nonpolar residues comprise the following: X1,X2, X77 to X78, X79, X80 to X81, X82, X83 to X88.

In some embodiments, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to higher temperatures isaccomplished by replacing ionic residues near the interface between thetwo monomers which do not apparently form polar/ionic contacts with theother monomer in SEQ ID NO:457 with more polar residues to stabilize theprotein. Polar or ionic residues participating in interactions at theinterface between the two monomers that can be replaced with more polarresidues comprise the following: X26 to X27.

In some of embodiments, shifting the threshold temperature Tbs ofglobular temperature sensitive bioswitch dimer to higher temperatures isaccomplished by minimizing entropy of the folded structure by replacinggeometrically constrained residues with smaller residues containing lessdegrees of freedom while maintaining the energetic contribution of thepolar contacts.

In some embodiments, a geometrically constrained residues in theN-terminal DNA binding domain can be replaced with a smaller residuecontaining less degrees of freedom while maintaining the energeticcontribution of the polar contacts For example, in TcI, residue K6 canbe mutated to N to shift the threshold temperature (FIG. 7J).

In some of embodiments, shifting the threshold temperature to highertemperatures is accomplished by replacing by replacing hydrophobic(nonpolar) residues at the dimerization interface with other hydrophobic(nonpolar) variants that optimize van-der-Waals contact area. Nonpolarresidues at the interface between the two monomers that can be replacedwith other hydrophobic (nonpolar) variants that optimize van-der-Waalscontact area comprise the following: X25, X34 to X37, X47. Polar,nonpolar or ionic residues at the interface between the two monomersthat can be replaced with other hydrophobic (nonpolar) variants thatoptimize van-der-Waals contact area comprise the following: X31 to X32,X48 to X52. Polar or ionic residues at the interface between twomonomers that can be replaced with other hydrophobic (nonpolar) variantsthat optimize van-der-Waals contact area comprise the following: X89 toX96. Polar or non-polar residues at the interface between two monomersthat can be replaced with other hydrophobic (nonpolar) variants thatoptimize van-der-Waals contact area comprise the following: X20 to X22,X28 to X30, X97 to X100, X104 to X105.

Alternatively, shifting the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer to higher temperatures isaccomplished by replacing residues surrounding the dimerizationinterface to charge-or-polarity-complemented pairs. Polar or ionicresidues participating in interactions at the interface between the twomonomers that can be replaced with charge- or polarity-complementedpairs comprise the following: X26 to X27.

In some embodiments, the threshold temperature Tbs of globulartemperature sensitive bioswitch dimer can be increased or decreased byreplacing in at least one monomer protein of the two monomer proteinsforming the temperature sensing domain any amino acid residues of SEQ IDNO: 457 and in particular of SEQ ID NO: 459 to have a substitutionselected using ΔΔG of substitution and/or ΔΔG of folding of the Rosettamodeling software that increases or decreases the related Tm andtherefore the Tbs as herein described.

In some embodiments of the globular temperature sensing domain, eachmonomer protein can have a globular temperature sensing amino acidsequence TTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVGKVIASQWPEETFG (SEQ ID NO: 459), or avariant of SEQ ID NO: 457 in which any of the amino acid residues of SEQID NO: 459 is substituted with a ΔΔG of substitution greater than 0.5Rosetta Energy Unit (REU) or lower than −0.5 Rosetta Energy Unit(R.E.U).

In some embodiments of the globular temperature sensing domain, eachmonomer protein can have a variant of the globular temperature sensingamino acid sequenceTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVGKVIASQWPEETFG (SEQ ID NO: 459), in which oneor more amino acid substitutions decreases the ΔG by >0.5-10, 0.5-5 orpreferably 1-4 of Rosetta energy units (REU), thus leading to a variantwith upshifted Tm. In such scenario, the ΔΔG has a negative value in arange from −0.5 to −10, or −0.5 to −5, or −1 to −4.

In some embodiments of the globular temperature sensing domain, eachmonomer protein can have a variant of the globular temperature sensingamino acid sequenceTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVGKVIASQWPEETFG (SEQ ID NO: 459), in which oneor more amino acid substitution increases the ΔG by >0.5-10, 0.5-5 orpreferably 1-4 of Rosetta energy units (REU), thus leading to a variantwith downshifted Tm. In such scenario, the ΔΔG has a positive value in arange from 0.5 to 10, or 0.5 to 5, or 1 to 4.

In particular, variants of globular temperature sensitive transcriptionfactors, having a globular temperature sensing amino acid sequence SEQID NO: 457 and in particular of SEQ ID NO: 459 can be performed bypredicting the possible mutations in the globular temperature sensingdomain interface that are stabilizing or neutral, and predicting theenergy of all possible mutations in the amino acid sequence SEQ ID NO:457 and in particular of SEQ ID NO: 459.

In some embodiments herein described, predicting the possible mutationsin the globular temperature sensing domain interface can be performed byrunning the Rosetta interface plasticity prediction as described above(application 3 in Lauck et al. (2010) [27] and selecting for allmutations predicted with frequency >0, consider these mutationsstabilizing or neutral. In particular, running Rosetta interfaceplasticity prediction can be performed with the following Inputs:Crystal structure (for example, PDB code 3BDN), Interacting chain IDs(chain a and chain b), Residues which are interacting to obtain thefollowing outputs: Sequences of variants which are predicted to functionand relative frequencies of variants which are predicted to function.

In some embodiments herein described, predicting the energy of allpossible mutations in the amino acid sequence SEQ ID NO: 457 and inparticular of SEQ ID NO: 459 can be performed by running the Rosettapoint mutation modeling prediction (application I in Lauck et al. (2010)[27] for the predicted possible mutations, then determine the change inR.E.U. (ΔΔG) and select the variants with associated R.E.U. rangesherein described.

The thermal transcriptional bioswitch herein described can encompassother proteins that operate on similar principles as TlpA or TcI. Theseinclude highly homologous proteins, such as the Coiled coil DNA bindingprotein KfrA, and engineered constructs such as a previously reportedsynthetic protein in which the Lambda cI binding domain is grafted ontothe GCN4 coiled coil [32].

In some embodiments, a WT TcI has a threshold temperature of about 40°C. and mutating M1V, L65S, K68R, F115L, D126G, D188G in TcI (“TcI₃₈”)generates a bioswitch with a threshold transcriptional activationcentered at 38° C. In another embodiment, mutating K6N, S33T, Y61H,L119P, F122C (“TcI₄₂”), generates another bioswitch with a thresholdtranscriptional activation centered at 42° C. (see Examples 1-2).

Coiled coil and globular thermal transcriptional bioswitch hereindescribed can encompass other proteins that operate on similarprinciples as TlpA or TcI. These include proteins highly homologous toTlpA or TcI, such as the coiled coil DNA binding protein KfrA, andengineered constructs such as a previously reported synthetic protein inwhich the Lambda cI binding domain is grafted onto the GCN4 coiled coil[32].

In some embodiments, the thermal bioswitches comprising coiled coiltranscriptional bioswitch dimers such as TlpA and/or globulartranscriptional bioswitch dimers such as TcI repressor families hereindescribed can be used to activate or inactivate gene expression in thebiomedically relevant range of 32 to 46° C. while demonstrating adynamic range similar to that of the wild-type protein (Table 1). Inparticular, transcriptional bioswitch dimers can be provided based onTlpA or other coiled coil transcriptional bioswitch dimer, or TcI orother globular transcriptional bioswitch dimers, that have a sharpthermal transition resulting in a large change in activityi.e. >100-fold over a few degrees, and a switching temperature Tbstunable to enable a broad range of applications. In addition, thebioswitches are orthogonal to endogenous cellular machinery andcompatible with other thermos-responsive components to allow multiplexedthermal logic.

In particular, in some embodiments, the temperature sensitivetranscription factors herein described, and in particular the coiledcoil and/or globular transcriptional bioswitch dimers herein described,can be integrated into thermal logic circuits to control multiplefunctions at different desired temperatures or confine activity towithin a narrow thermal range.

The term “logic circuit”, “genetic circuit” or “circuit,” as used hereinindicates a collection of molecular components (herein also indicated asnodes) connected one to another by biochemical reactions according to acircuit design. In particular, in a genetic circuit the molecularcomponents are connected one to another by the biochemical reactions sothat the collection of molecular components is capable to provide aspecific output in response to one or more inputs. The term biochemicalreactions as used herein comprise

The term “molecular component” or “node” as used herein in connectionwith the genetic circuit indicates a chemical compound comprised in acellular environment. Exemplary molecular components thus comprisepolynucleotides, such as ribonucleic acids or deoxyribonucleic acids,polypeptides, amino acids, and/or other small or large molecules and/orpolymers that can be found in a cellular environment.

In genetic circuits in the sense of the present disclosure, themolecular components forming parts of the genetic circuit are geneticmolecular components. The term “genetic molecular component” as usedherein indicates a molecular unit formed by a gene, an RNA transcribedfrom the gene or a portion thereof and optionally a protein translatedfrom the transcribed RNA. In genetic circuits herein described, thebiochemical reactions connecting the genetic molecular component toanother molecular component of the circuit can involve any one of thegene, the transcribed RNA and/or the polypeptide forming the molecularcomponent.

A gene comprised in a genetic molecular component is a polynucleotidethat can be transcribed to provide an RNA and typically comprises codingregions as well as one or more regulatory sequence regions which is asegment of a nucleic acid molecule which is capable of increasing ordecreasing transcription or translation of the gene within an organismeither in vitro or in vivo. In particular, coding regions of a geneherein described can comprise one or more protein coding regions whichwhen transcribed and translated produce a polypeptide, or if an RNA isthe final product, only a functional RNA sequence that is not meant tobe translated. Regulatory regions of a gene herein described comprisepromoters, transcription factor binding sites, operators, activatorbinding sites, repressor binding sites, enhancers, protein-proteinbinding domains, RNA binding domains, DNA binding domains, silencers,insulators and additional regulatory regions that can alter geneexpression in response to developmental and/or external stimuli as willbe recognized by a person skilled in the art.

A protein comprised in a molecular component can be proteins withactivating, inhibiting, binding, converting, or reporting functions.Proteins that have activating or inhibiting functions typically act onoperator sites encoded on DNA, but can also act on other molecularcomponents. Proteins that have binding functions typically act on otherproteins, but can also act on other molecular components. Proteins thathave converting functions typically act on small molecules, and convertsmall molecules from one small molecule to another by conducting achemical or enzymatic reaction. Proteins with converting functions canalso act on other molecular components. Proteins with reportingfunctions have the ability to be easily detectable by commonly useddetection methods (absorbance, fluorescence, for example), or otherwisecause a reaction on another molecular component that causes easydetection by a secondary assay (eg. adjusts the level of a metabolitethat can then be assayed for). The activating, inhibiting binding,converting, or reporting functions of a protein typically form theinteractions between genetic components of a circuit. Exemplary proteinsthat can be comprised in a genetic molecular component comprisemonomeric proteins and multimeric proteins, proteins with tertiarty orquaternary structure, proteins with linkers, proteins with non-naturalamino acids, proteins with different binding domains, and other proteinsknown to those skilled in the art. Specific exemplary proteins includeTetR, LacI, LambdaCI, PhlF, SrpR, QacI, BetR, LmrA, AmeR, LitR, met,AraC, LasR, LuxR, IpgC, MxiE, Gal4, GCN4, GR, SP1, CREB, and othersknown to a skilled person in the art.

In some embodiments of genetic circuits herein described, one or moremolecular components is a recombinant molecular component that can beprovided by genetic recombination (such as molecular cloning) and/orchemical synthesis to bring together molecules or related portions frommultiple sources, thus creating molecular components that would nototherwise be found in a single source.

In embodiments of the thermal logic circuits herein described, a thermallogic circuit comprises one or more genetic molecular component,connected one to another in accordance to a circuit design byactivating, inhibiting, binding or converting reactions to form a fullyconnected network of interacting components. in which at least onegenetic molecular component is under control of a temperature sensitivetranscription factor herein described.

The term “activating” as used herein in connection with a molecularcomponent of a genetic circuit refers to a reaction involving themolecular component which results in an increased presence of the sameor a different molecular component in the cellular environment. Forexample, activation of a genetic molecular component indicates one ormore reactions involving the gene, RNA and/or protein of the geneticmolecular component resulting in an increased presence of the gene, RNAand/or protein of the same or a different genetic molecular component(e.g. by increased expression of the gene of the molecular component,and/or an increased translation of the RNA). An example of activation ofa genetic molecular component of a genetic circuit comprises theexpression of a target gene upon exposure to a specific range oftemperatures.

The term “inhibiting” as used herein in connection with a molecularcomponent of a genetic circuit refers to a reaction involving themolecular component of the genetic circuit and resulting in a decreasedpresence of the same or a different molecular component in the cellularenvironment. For example, inhibition of a genetic molecular componentindicates one or more reactions involving the gene, RNA and/or proteinof the genetic molecular component resulting in a decreased presence ofthe same or a different gene, RNA and/or protein (e.g. by decreasedexpression of the gene of the molecular component, and/or a decreasedtranslation of the RNA). An example of inhibition of a genetic molecularcomponent of a genetic circuit comprises the reaction of a repressingprotein (eg. TlpA or TcI) that reduces the expression of a genecontrolled by a TlpA or TcI promoter.

The term “binding” as used herein in connection with molecularcomponents of a genetic circuit refers to the connecting or uniting twoor more molecular components of the circuit by a bond, link, force ortie in order to keep two or more molecular components together, whichencompasses either direct or indirect binding where, for example, afirst molecular component is directly bound to a second molecularcomponent, or one or more intermediate molecules are disposed betweenthe first molecular component and the second molecular component anothermolecular component of the circuit. Exemplary bonds comprise covalentbond, ionic bond, van der waals interactions and other bondsidentifiable by a skilled person. In some embodiments, the binding canbe direct, such as the production of a polypeptide scaffold thatdirectly binds to a scaffold-binding element of a protein. In otherembodiments, the binding can be indirect, such as the co-localization ofmultiple protein elements on one scaffold. In some instances binding ofa molecular component with another molecular component can result insequestering the molecular component, thus providing a type ofinhibition of said molecular component. In some instances binding of amolecular component with another molecular component can change theconformation or function of the molecular component, as in the case ofallosteric interactions between proteins or dimerization between twomonomers, thus providing a type of activation or inhibition of the boundcomponent.

In embodiments of a genetic circuit, the molecular components areconnected one with another according to a circuit design in which amolecular component is an input and another molecular component is anoutput. In particular, a genetic circuit typically has one or more inputor start molecular component which activates, inhibits, and/or bindsanother molecular component, one or more output or end molecularcomponent which are activated, inhibited, bound and/or converted byanother molecular component, and intermediary molecular components eachinhibiting, binding and/or converting another molecular component andbeing activated, inhibited, bound and/or converted by another molecularcomponent.

In embodiments herein described, the molecular components are typicallyconnected together according to the circuit design in defined patternsof interactions between components called circuit motif. A circuit motiftypically has inputs and outputs and performs an information processingfunction that is one level higher than recombinant genetic components.

Exemplary circuit motifs that can be used to connect collections ofmolecular components in a genetic circuit according to a circuit designcomprise a feed-forward loop (wherein the output is a pulse) [33], anoscillator (wherein the output is an oscillatory output) [34], arepression cascade (wherein the output is repression of the expressionof a molecular component of the circuit) [35], a switch (wherein theoutput is expression of either one molecular component of the circuit inresponse to one input or another molecular component of the circuit inresponse to another input) [36] (see Examples 2-4 and FIGS. 8A-8H), or acascade (wherein the circuit transmits the input signal to an outputsignal) [37], and other circuit designs which will be identifiable by askilled person upon reading of the present disclosure.

Genetic circuits in the sense of the disclosure can comprise more thanone circuit motif and in particular two or more circuit motifs as willbe understood by a skilled person.

In some embodiments, the transcriptional activity of a transcriptionregulatory factor can be modified by molecular modifications made to aprotein that change its conformation and affects the activity of theprotein as it pertains to inhibition, activation, sequestration, actingon a substrate or other biomolecular functions; mutations to proteins inwhich a molecular component binds to a protein and changes itsconformation or dimerization state resulting in a change of activity aswill be understood by a person skilled in the art.

In some embodiments, the transcription regulatory factors of the thermallogic circuit are thermal transcriptional bioswitches herein describedcomprising a DNA-binding domain and a temperature-sensing domain. Uponexposure to a specific temperature range, the temperature sensing domaincan cooperatively modulate the assembly of two polypeptide chains eachhaving a globular structure or single-stranded coil into a functionaldimer. The correctly configured dimer can then bind to the a promotersequence and represses the transcription output of the system either byoccluding the binding site for RNA polymerase or by impeding the 5′ to3′ motion of the RNA polymerase along the DNA.

In the genetic circuits herein described at least one molecularcomponent of the circuit can be a reportable molecular componentdetectable in a cell-free system and/or in a target environment when thegenetic circuit operates according to the circuit design.

The term “reportable molecular component” as used herein indicates amolecular component capable of detection in one or more systems and/orenvironments. The terms “detect” or “detection” as used herein indicatesthe determination of the existence, presence or fact of a target in alimited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate. The “detect” or“detection” as used herein can comprise determination of chemical and/orbiological properties of the target, including but not limited toability to interact, and in particular bind, other compounds, ability toactivate another compound and additional properties identifiable by askilled person upon reading of the present disclosure. The detection canbe quantitative or qualitative. A detection is “quantitative” when itrefers, relates to, or involves the measurement of quantity or amount ofthe target or signal (also referred as quantitation), which includes butis not limited to any analysis designed to determine the amounts orproportions of the target or signal. A detection is “qualitative” whenit refers, relates to, or involves identification of a quality or kindof the target or signal in terms of relative abundance to another targetor signal, which is not quantified.

In some embodiments of the genetic circuit according to the disclosure,the reportable molecular component can be a molecular component linkedor comprising a label wherein the term label refers to a compoundcapable of emitting a labeling signal, including but not limited toradioactive isotopes, fluorophores, chemiluminescent dyes, chromophores,enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes,metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin,streptavidin or haptens) and the like. The term “fluorophore” refers toa substance or a portion thereof which is capable of exhibitingfluorescence in a detectable image as seen for fluorescent reporter GFPand RFP in Examples 2-4. As a consequence, the wording “labeling signal”as used herein indicates the signal emitted from the label that allowsdetection of the label, including but not limited to radioactivity,fluorescence, chemiluminescence and the like.

Accordingly, in genetic circuit of the disclosure, operativeness of thegenetic circuit can be detected by detecting the reportable molecularcomponent of the thermal logic circuit in vivo or in vitro when thegenetic circuit operates according to the circuit design.

In embodiments, herein described, temperature sensitive genetic circuitcomprise at least one genetic molecular component under regulation of atleast one of the coiled coil and/or globular transcription bioswitchdimers herein described and/or of at least one of other temperaturesensitive transcription factors known or identifiable by a skilledperson.

Additional temperature sensitive or thermal genetic circuit are alsodescribed herein in which at least one genetic molecular component isunder regulation of at least one of the coiled coil and/or globulartemperature sensitive transcription factors herein described and/or ofat least one of other temperature sensitive transcription factors knownor identifiable by a skilled person.

Exemplary other temperature switches known or identifiable by a skilledperson comprise cI mutant from Phage L1 (bioswitch temperature isbetween 35-38° C.; globular) [38], cI mutant from Phage P1 (bioswitchtemperature is ˜40° C.; globular) [39], c repressor from Phage Mu(bioswitch temperature is between 30° C.-42° C.) [40], RheA (bioswitchtemperature between 37° C.-41° C.; note: is a dimer, but switching doesnot seem to be caused by conversion to monomer) [41], GmaR (bioswitchtemperature between 22° C.-34° C.; structure is alpha-helical/random)[42], Temperature Sensitive LacI variants: Gly187Ser (bioswitchtemperature is 42° C.), Ala241Thr (bioswitch temperature is 40° C.),Gly265Asp (bioswitch temperature is 37° C.); alpha-helical C-terminaltetramerization domain) [43]. Temperature Sensitive TetR variants: HightetR expressors of G21E (bioswitch temperature is between 28° C. and 37°C.), A89D (bioswitch temperature is ˜37° C.), I193N (bioswitchtemperature is between 37° C.-42° C.). Low tetR expressors wererepressed at all temperatures. [44], and RovA (bioswitch temperature isbetween 25° C. and 37° C.; structure is alpha-helical/beta-sheet/random)[45]. Additional temperature sensitive switches capable of being used intemperature sensitive genetic circuit herein described, are identifiableby a skilled person.

In some embodiments of thermal genetic circuits or temperature sensitivegenetic circuits herein described, molecular components can be connectedto form one or more circuit motifs within the thermal genetic circuitand at least one component of at least one circuit motifs is configuredto be under regulation of a temperature sensitive transcription factorherein described.

In some embodiments, the thermal circuit can comprise one circuit motifthat further comprises a thermal transcriptional bioswitch hereindescribed, a promoter, and a reportable molecular component as shown inthe example of FIG. 2A. The thermal transcriptional bioswitch can be acoiled coil temperature sensitive transcriptional factor such as TlpAand/or a variant thereof, and/or globular temperature sensitivetranscriptional factors such as TcI and/or a variant thereof, or othertemperature sensitive transcriptional factors that can be identified bya person of ordinary skill in the art. The promoter can be a heat-shockpromoter such as pTSR, pHSP, pR-pL, GrpE, HtpG, Lon, RpoH, Clp, DnaK andother heat-shock promoters as will be understood by a skilled person inthe art. The reportable molecular component can be a fluorescentreporter such as GFP and RFP.

In some embodiments, thermal genetic circuits of the disclosure can bedesigned to comprise two or more circuit motifs, each motif comprising athermal transcriptional bioswitch herein described, a promoter, and areportable molecular component, in which the thermal transcriptionalbioswitch can be a coiled coil temperature sensitive transcriptionalfactor such as TlpA and/or a variant therefore and/or globulartemperature sensitive transcriptional factors such as TcI and/or avariant thereof or other temperature sensitive transcriptional factorsthat can be identified by a person of ordinary skill in the art.

In some embodiments, one or more molecular components of the thermalgenetic circuits herein described can be a temperature sensitivetranscription factor such as a coiled coil transcriptional bioswitchdimers such as TlpA and/or a variant thereof and/or globulartranscriptional bioswitch dimer such as TcI and/or a variant thereof.For example, temperature sensitive transcription factors hereindescribed can be used in a temperature sensitive genetic circuit to beoperated in a target environment at at least two target temperatures.The temperature sensitive genetic circuit comprises one or moremolecular components connected one to another by biochemical reactionsaccording to a circuit design. In the temperature sensitive geneticcircuit, at least one of the molecular components is a temperaturesensitive genetic molecular components comprising a coiled coil and/orglobular temperature sensitive transcription factor herein describedhaving a bioswitch temperature Tbs in the target environment equal toone of the at least two target temperatures. In the temperaturesensitive genetic circuit, each of the temperature sensitive geneticmolecular component is configured to activate or inhibit another geneticmolecular component of the genetic circuit at a temperature sensitivemolecular component bioswitch temperature equal to the bioswitchtemperature Tbs of the coiled coil and/or globular temperature sensitivetranscription factor.

In particular, in some embodiments temperature sensitive geneticcircuits comprising thermal transcription bioswitches possibly includingcoiled coil or globular temperature sensitive transcription factorsherein described can operate at a target environment temperature equalto a bioswitch temperature Tbs from 20 to 39° C.

In particular in some embodiments, in a temperature sensitive geneticcircuit herein described, at least one of the temperature sensitivegenetic molecular components of the genetic circuit is configured toactivate or inhibit another molecular component of the genetic circuitat a temperature sensitive molecular component bioswitch temperatureequal to the bioswitch temperature Tbs 20 to 39° C.

In some embodiments, in a temperature sensitive genetic circuit hereindescribed, at least one of the temperature sensitive genetic molecularcomponents of the genetic circuit is configured to activate or inhibitanother molecular component of the genetic circuit at a temperaturesensitive molecular component bioswitch temperature equal to thebioswitch temperature Tbs from 32 to 39° C.

In some embodiments, in a temperature sensitive genetic circuit hereindescribed, at least one of the temperature sensitive genetic molecularcomponents of the genetic circuit is configured to activate or inhibitanother molecular component of the genetic circuit at a temperaturesensitive molecular component bioswitch temperature equal to thebioswitch temperature Tbs from 41 to 43° C.

In some embodiments, in a temperature sensitive genetic circuit hereindescribed, at least one of the temperature sensitive genetic molecularcomponents of the genetic circuit is configured to activate or inhibitanother molecular component of the genetic circuit at a temperaturesensitive molecular component bioswitch temperature equal to thebioswitch temperature Tbs from 45 to 80° C.

For example, as shown in the Examples section, the thermal geneticcircuit comprising a thermal transcriptional bioswitch of TlpA WT,variants TlpA36 and TlpA39 can be turned on at temperatures of 43° C.,36° C., and 39° C. respectively.

In some embodiments, a temperature sensitive multiplexed genetic circuitcan be designed to be operated in a target environment at at least twodifferent target temperatures. The temperature sensitive multiplexedgenetic circuit comprises one or more molecular components connected oneto another by biochemical reactions according to a circuit design. Inthe multiplexed genetic circuit at least two genetic molecularcomponents are temperature sensitive molecular components, eachconfigured to activate or inhibit another molecular component of thegenetic circuit at a temperature sensitive molecular component bioswitchtemperature.

In the multiplexed temperature sensitive genetic circuit, at least onefirst temperature sensitive molecular component is configured toactivate or inhibiting a first molecular component at a first bioswitchtemperature Tbs₁, and at least one second temperature sensitivemolecular component configured to activate or inhibit a second anothermolecular component at a second bioswitch temperature Tbs₂. In themultiplexed genetic circuit the first bioswitch temperature Tbs₁ isequal to one of the at least two different target temperatures of thetarget environment and the second bioswitch temperature Tbs₂ is equal toanother one of the at least two different target temperatures of thetarget environment. In the multiplexed genetic circuit, the firstanother molecular component is different from the second anothermolecular component and the first bioswitch temperature Tbs₁ isdifferent from the second bioswitch temperature Tbs₂. In someembodiments at least one of the temperature sensitive genetic molecularcomponents comprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs₁, or Tbs₂.

In some of these embodiments, the thermal genetic circuits can compriseat least two circuit motifs to form a multiplexed genetic circuit inwhich at least two signals generated from the at least two circuitmotifs are activated or emitted at two different temperatures and thencombined into one signal upon their activation in a shared environment,e.g. a cell environment. One of the circuit motifs can comprise a firsttemperature sensitive transcriptional factor that activates at a firsttemperature Tbs1, while the other circuit motif can comprise a secondtemperature sensitive transcriptional factor that activates at a secondtemperature Tbs2, the second temperature being different from the firsttemperature.

In the temperature sensitive multiplexed genetic circuit hereindescribed, the first temperature at which the first temperaturesensitive transcriptional factor activates can be a temperature Tbs1selected between 20° C. and 39° C., or 32 to 39° C. or 41 to 43° C., or45 to 80° C. and the second temperature at which the second temperaturesensitive transcriptional factor activates can be a temperature Tbs2selected between 20° C. and 39° C., or 32 to 39° C. or 41 to 43° C., or45 to 80° C.

For example, as shown in FIGS. 8A-8D (Example 4), the multiplexedgenetic circuit comprises a first circuit motif having TlpA36 variant asthe thermal transcriptional bioswitch, pTlpA as the promoter and GFP asthe reportable molecular component, the first circuit motif beingactivated at a temperature of 36° C. The multiplexed genetic circuit ofFIGS. 8A-8D further comprises a second circuit motif having TcI WT asthe thermal transcriptional bioswtich, pR-pL as the promoter and RFP asthe reportable molecular component, the second circuit motif beingactivated at a temperature higher than the activation temperature of thefirst bioswitch (about 40° C.). Thus, upon activation of bothbioswitches (for example, at 42° C. or above), the medium shows bothgreen and red fluorescence.

In some embodiments, a temperature sensitive bandpass filter can bedesigned to be operated in a target environment at least two targettemperatures forming a target temperature range, The bandpass filter isconfigured to be operated within genetic circuit comprising one or moremolecular components connected one to another by biochemical reactionsaccording to a circuit design. The temperature sensitive bandpass filtercomprises a first temperature sensitive genetic molecular componentsconfigured to activate a first another molecular component of thegenetic circuit at a first bioswitch temperature Tbs1 and to inhibit thefirst another genetic molecular component at a second bioswitchtemperature Tbs2. The temperature sensitive bandpass filter furthercomprises a second temperature sensitive genetic molecular componentsconfigured to inhibit the first genetic molecular component and activateor inhibit a second another molecular component of the genetic circuitat the bioswitch temperature Tbs2.

In the temperature sensitive bandpass filter, the first bioswitchtemperature Tbs₁ is equal to one of the at least two different targettemperatures of the target environment, the second bioswitch temperatureTbs₂ is equal to another one of the at least two different targettemperatures of the target environment In the multiplexed geneticcircuit, the first another molecular component is different from thesecond another molecular component and the first bioswitch temperatureTbs₁, is different from the second bioswitch temperature Tbs₂. In someembodiments at least one of the temperature sensitive genetic molecularcomponents comprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs₁, and/or Tbs₂.

In some other embodiments, the thermal genetic circuits are designed tocomprise at least two circuit motifs to form a thermal or temperaturesensitive bandpass filter in which a first bioswitch of a first circuitmotif activates at a first temperature Tbs 1 and deactivates at a secondtemperature Tbs 2 and at the second temperature Tbs 2 a second bioswitchof a second circuit motif activates another component and deactivatesthe first bioswitch. Consequently, the two signals generated from thetwo circuit motifs alternate within a certain temperature range formedby the at least two target temperatures.

In particular, the first circuit motif comprises at least onetemperature sensitive transcriptional factor that activates at a firsttemperature Tbs 1 and deactivates at a second temperature Tbs 2 and thesecond circuit motif comprises at least one temperature sensitivetranscriptional factor that activates at the second temperature Tbs2,the first temperature being different from the second temperatures.

In some embodiments, the at least one temperature sensitivetranscriptional factor of the first circuit motif activates at a firsttemperature Tbs 1 selected between 20° C. and 39° C., or 32 to 39° C. or41 to 43° C., or 45 to 80° C. and deactivates at a second temperatureTbs 2 selected between 20° C. and 39° C. or 32 to 39° C. or 41 to 43°C., or 45 to 80° C. and the at least one temperature sensitivetranscriptional factor of the second circuit motif activates at thesecond.

For example, as shown in FIGS. 8E-8H (Example 4), the thermal bandpassfilter comprises a first circuit motif having TlpA WT as the thermaltranscriptional bioswitch, pTlpA as the operator-promoter and GFP as thereportable molecular component, the first circuit motif being activatedat a temperature of 44° C. The thermal bandpass filter further comprisesa second circuit motif having TcI38 and the wild type cI repressor asthe thermal transcriptional bioswitches, pR-pL as the operator-promoterand RFP as the reportable molecular component, the second circuit motifbeing activated at a temperature of 38° C. and deactivated at atemperature of 44° C. (see FIG. 8G).

In embodiments herein described, the thermal bio switches integrated inmultiplexed and/or bandpass thermal genetic circuits can comprise one ormore coiled coil temperature sensitive transcriptional factor such asTlpA or variants thereof, one or more globular temperature sensitivetranscriptional factors such as cI, TcI and variants thereof, and/or oneor more other thermal transcriptional factors identifiable to a personof ordinary skill in the art which are orthogonal to one or moremolecular components of the target environment and/or the circuits.

In particular, in several embodiments of thermal genetic circuits, thethermal transcriptional bioswitches used herein in the thermal geneticcircuits are orthogonal to endogenous cellular circuits and compatiblewith other thermo-responsive components in the target cell environment.In several embodiments, the thermal transcriptional bioswitches usedherein in combination in the thermal genetic circuits such as bandpassand/or multiplexed genetic circuits are orthogonal one with respect tothe other.

Temperature sensitive bioswitches herein described can be included inone or more vectors and in particular expression vectors. The term“vector” indicates a molecule configured to be used as a vehicle toartificially carry foreign genetic material into a cell, where it can bereplicated and/or expressed. An expression vector is configured to carryand express the material in a cell under appropriate conditions.

Vectors herein described can comprise a polynucleotide encoding for oneor more coiled coil and/or globular temperature sensitive transcriptionfactors herein described, under control of one or more regulatorysequence regions in a configuration allowing to express the temperaturesensitive transcription factors encoded by the polynucleotide inpresence of suitable cellular transcription and translation factors.

In some embodiments an expression vector can comprise one or morepolynucleotides encoding one or more temperature sensitive transcriptionfactors herein described under control of one or more regulatorysequences including promoter and/or enhancer regions in a configurationallowing regulation of expression of the one or more temperaturesensitive dimer proteins encoded by the polynucleotide in presence ofnecessary cellular transcription and translation factors. The regulatorysequences such as promoter and/or enhancer regions can be arrangedproximally and/or distally 5′ and/or 3′ to the one or morepolynucleotides encoding for a temperature sensitive transcriptionfactors herein described. The expression vector can also compriseadditional regulatory elements such as ribosome binding sites, andtranscription termination sequences. In some embodiments, the regulatorysequences of promoter and/or enhancer regions regulating expression ofone or more polynucleotides encoding a temperature sensitivetranscription factors comprise DNA regulatory region regulated bybinding of one or more temperature sensitive transcription factors.

In some embodiment, one or more vectors in combination can furthercomprise a target DNA polynucleotide controlled by one or moretemperature sensitive bioswitches herein described (herein alsoindicated as gene or target gene) and the related DNA regulatoryregions. In particular one or more target genes can be comprised in avector, which can be the same vector or a different vector from a vectorencoding a polynucleotide encoding a temperature sensitive dimerprotein. The one or more target genes are comprised in the vector in aconfiguration allowing regulation of expression of the one or moretarget genes in presence of a corresponding temperature sensitivebioswitch herein described as well as necessary cellular transcriptionand translation factors. The regulatory sequences such as promoterand/or enhancer regions can be arranged proximally and/or distally 5′and/or 3′ to the one or more target genes and includes DNA regulatoryregion corresponding to e temperature sensitive transcription factors.In some embodiments, the vectors can comprise regulatory sequences ofpromoter and/or enhancer regions regulating expression of one or morepolynucleotides encoding for non-thermally regulated molecularcomponents which can be comprised in a genetic circuit.

In some embodiments, one or more vectors are described containingmolecular components of any one of the thermal genetic circuits isdescribed. The vector can be designed to convert a cell into atemperature-dependent cell wherein one or more biological properties areactivated in a temperature sensitive manner by one or more temperaturesensitive genetic circuit herein described. The vector can be a plasmid,a virus such as a bacteriophage, or a bacterium that is designed todeliver DNA to another bacterial species via conjugation.

In some embodiments, a DNA or RNA molecule containing one or more thethermal genetic circuits is described. The DNA or RNA molecule can beused to create the vectors described above or to transform bacterialcells.

In some embodiments, vectors can be used to create a temperaturesensitive cell to be operated in a target environment at at least twotarget temperatures. The temperature sensitive cell comprises atemperature sensitive genetic circuit herein described. In thetemperature sensitive genetic circuit, at least one of the geneticmolecular components is a temperature sensitive genetic molecularcomponents, each having a bioswitch temperature Tbs_(C) in the cellequal to at least one of the at least two target temperatures. In someembodiments at least one of the temperature sensitive genetic molecularcomponents comprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs_(C)

In some embodiments, the thermal logic circuits with the integratedbioswitches herein described can be used in in vivo microbial therapyscenarios, including spatially precise activation using focusedultrasound, modulation of activity in response to a host fever, andself-destruction after fecal elimination to prevent environmental escapeand provide a capability for coupling endogenous or applied thermalsignals to cellular function in biomedical and industrial applications.

In some embodiments, a temperature-sensitive therapeutic cell isdescribed. The temperature-responsive therapeutic cell comprises atemperature sensitive genetic circuit herein described comprising atleast one temperature sensitive molecular component and at least onetherapeutic molecular component and designed to provide a therapeuticoutput. In the genetic circuit of the therapeutic cell, the at least onetemperature sensitive molecular components is configured to activate orinhibit the at least one therapeutic molecular component at atherapeutic bioswitch temperature Tbs_(T) to provide the therapeuticoutput of the genetic circuit.

A therapeutic circuit output in the sense of the disclosure comprisesexpression of one or more therapeutic proteins that are secreted ordisplayed on the cell membrane. A therapeutic protein so expressed caninteract under appropriate conditions with surrounding cells ormolecules of the host in a way that alters the biological state of thehost, leading to effects such as the death of certain host cells, changein the state of certain host cells (for example, immune cell activationor inactivation, stem cell differentiation or neuronal excitabilityincrease or decrease), recruitment of certain host cells (such as immunecells). Examples of therapeutic genes include Cytokines to modulate theimmune response, such as IL-10, IL-2, TNF, TGFbeta, Cell-killingproteins such as cytolysin, hemolysin, growth factors such as VEGF orBDNF. The system can control the expression of one or more genesassembled in series.

In some embodiments bioswitch temperature Tbs_(T) is achieved in thetemperature sensitive therapeutic cell in response to a thermalstimulus. In some embodiments, the thermal stimulus is selected from ahost fever or external source of thermal energy such as focusedultrasound, infrared, magnetic particle hyperthermia. In someembodiments at least one of the temperature sensitive genetic molecularcomponents comprises a coiled coil and/or globular temperature sensitivetranscription factor herein described having a bioswitch temperature Tbsequal to Tbs_(T).

The temperature-responsive therapeutic cells can comprise a plurality ofbacterial cells such as E. coli, Salmonella, Bacteroides, Lactobacillus.The cells can be natural cells with desirable therapeutic propertiessuch as ability to colonize the GI tract or home to and colonize atumor, or they can be genetically modified cells that have beenengineered for enhanced disease homing, proliferation, and additionalproperties identifiable by a skilled person.

In some embodiments, the temperature sensitive therapeutic cells cancomprise a plurality of eukaryotic cells. In some embodiments, theeukaryotic cells are unicellular organisms. Exemplary unicellulareukaryotes comprise protozoa, such as ciliates such as Paramecia,Stentors and Vorticella, amoebas such as Physarum and Entamoeba,unicellular algae such as euglenophyta, chlorophyte, diatoms,dinoflagellates, unicellular fungi such as yeasts such as Saccharomycesand candida species. For example, in some embodiments, the temperaturesensitive therapeutic cells comprise genetically modified yeast cellsthat are introduced into an individual to secrete a therapeuticsubstance in a temperature-regulated manner. In some embodiments, thetemperature sensitive therapeutic cells are cells in or isolated frommulticellular eukaryotic species. Multicellular eukaryotic speciescomprise mammalian species such as animals, plants, and multicellularfungi. In some embodiments, multicellular eukaryotes comprise speciessuch as Homo sapiens and Mus musculus, for example, among others. Insome embodiments, temperature sensitive therapeutic cells comprise stemcells, progenitor cells, induced pluripotent stem cells, and othersidentifiable by a skilled person. In some embodiments, temperaturesensitive therapeutic cells comprise genetically engineered mammalianstem cells, for or example, genetically engineered stem cells that havebeen designed to secrete therapeutic substance after introduction into ahost, such as a human.

In some embodiments, the temperature-responsive therapeutic cells cancomprise a plurality of mammalian cells compatible with the target host,such as T-cells, hematopoietic stem cells, mesenchymal stem cells,neural precursor cells, macrophages, fibroblasts or cardiomyocytes.

The temperature sensitive therapeutic cells herein described can containa thermal genetic circuit, which includes at least one therapeutic genewhose transcription is controlled by one or more transcription controlelements that are regulated by temperature. These transcriptionalcontrol elements include at least one DNA sequence upstream of thetherapeutic gene (e.g. an operator or promoter) and a thermaltranscriptional bioswitch such as a temperature-dependent repressorprotein that binds to this operator. The thermal transcription bioswitchor temperature-dependent repressor protein has the property that changesof temperature around a certain desired set-point (e.g. 40° C.) causethe protein to unbind from the corresponding DNA operator and allow thegene it regulates to be expressed. The unbinding of the repressor causesgene expression because it either reveals a promoter binding site of theDNA that was blocked by the repressor or because it prevents the boundrepressor from stalling the RNA polymerase whose transcriptionalactivity is initiated upstream from the operator. The thermaltranscription bioswitch can be mutated from natural repressors so thattheir threshold temperature is different from the natural thresholdtemperature.

In some embodiments, the temperature sensitive therapeutic circuit canbe designed to include at least one temperature sensitivetranscriptional factor activating at a therapeutic temperature bioswitchTbs_(T) between 32 to 45° C., in particular between 32° C. and 39° C.,or between 32 to 36° C. or 36 to 39° C., or 39 to 41° C., or above 41°C. possibly between 41 to 43° C., or 41 to 45° C.

In some embodiments, the temperature-responsive therapeutic cells can bedesigned to activate the expression of their therapeutic gene inresponse to a thermal stimulus, such as ingestion into a host (set pointaround 36° C.), a host fever (set point around 38.5° C.) or heatingusing an external source of energy such as focused ultrasound, infrared,magnetic particle hyperthermia (see FIGS. 6C and 6D; FIGS. 9A-9E; FIGS.10A-10C).

Examples of therapeutic genes include Cytokines to modulate the immuneresponse, such as IL-10, IL-2, TNF, TGFbeta, Cell-killing proteins suchas cytolysin, hemolysin, growth factors such as VEGF or BDNF. The systemcan control the expression of one or more genes assembled in series.

In some embodiments of temperature-responsive therapeutic cells, ratherthan directly controlling the therapeutic genes, the thermal bioswitchcan control the expression of another protein that controls theexpression of those genes. For example, the thermal bioswitch controlsthe expression of a transcription factor that acts on a promoter thatdrives the expression of therapeutic genes. By selecting this promoterto be stronger or weaker, the magnitude of thermally induced expressionof therapeutic genes is controlled.

Alternatively, the thermal bioswitch can control the expression of arecombinase (e.g. FimB, FimE, TP091, Bxb1 or phiC31), which in turn actson a DNA sequence in which the therapeutic genes are configured suchthat the thermally triggered expression of recombinase causes them to bepermanently switched on. This allows the therapeutic cell to have apermanent response to a single thermal trigger. Conditional expressionof a second recombinase (e.g. under the control of a chemical inducer)can be used to reverse the action of the temperature-inducedrecombinase.

Alternatively, the thermal bioswitch can control the expression of agenome editing protein or guide RNA, such as Cas9 or one itsderivatives. The Cas9 can be active as a DNA-cutting enzyme or couldhave one of the other functions engineered for it, such as acting as arepressor or transcriptional activator (e.g. dCas9).

In some embodiments, in addition to placing the therapeutic genes,transcription factors or recombinases downstream of thetemperature-dependent repressor's operator sequence, we also have a copyof the temperature-sensitive repressor itself. This creates a negativefeedback loop such that if there is any leaky expression due to the lowconcentration of repressor protein, it will lead to a buildup in theconcentration of that protein. This is implemented in our examples (e.g.FIG. 2A and FIG. 8F) where the expression of the temperature-sensitiverepressor is driven by both, its own operator and a constitutivelyactive promoter (pLacI).

In some embodiments, genes downstream of the temperature-dependentrepressor's operator sequence include a reportable molecular componentgene designed to signal the activation of the circuit. This reportergene can be a fluorescent protein, a luminescent protein or a protein(or group of proteins) detectable by magnetic resonance imaging orultrasound. The reporter gene can also be a protein that results inlocal accumulation of a radiation emitting compound detectable bypositron emission tomography or single photon emission computedtomography.

In some embodiments, the operator is bi-directional (e.g. TlpA, seeFIGS. 4C-4E). and there are two genes placed under its control byputting one in one orientation and the other in the other orientation.

In some embodiments, the operator is a promoter that is activated whenthe temperature-dependent repressor is bound to it, rather than when itunbinds. The example is TcI with the pRM promoter [46].

The temperature-responsive therapeutic cells herein described cancontain these genetic circuits either in their genome or on a plasmid.The cells exhibit 100-fold difference in gene expression between the offstate and on state and achieve 10-fold switching over a temperaturerange smaller than 5° C.

In some embodiments, temperature-responsive biosynthetic cells aredescribed. The temperature-responsive biosynthetic cells comprise aplurality of bacterial cells such as E. coli or B. subtilis that adesigned for use in a biosynthetic pathway, e.g. in a bioreactor.Examples include cells engineered to catalyze a certain organic chemicalreaction, produce a biofuel, take up a contaminant for bioremediation,etc.

The temperature-responsive biosynthetic cell is designed to activate theexpression of its therapeutic gene in response to a change intemperature in the biosynthetic reaction apparatus, which is controlledby the reactor's operator (see FIGS. 6C and 6D). Examples ofbiosynthetic genes include enzyme in a biosynthetic pathway andregulatory protein that controls the activity of one or more enzymes.The system can be designed to control the expression of one gene or moregenes assembled in series.

In some embodiments, rather than directly controlling the therapeuticgenes, the system can control the expression of another protein thatcontrols the expression of those genes. For example, the thermalbioswitch is designed to control the expression of a transcriptionfactor that acts on a promoter that drives the expression of therapeuticgenes. By selecting this promoter to be stronger or weaker, themagnitude of thermally induced expression of biosynthetic genes iscontrolled.

Alternatively, the thermal bioswitch can control the expression of arecombinase, which in turn acts on a DNA sequence in which thebiosynthetic genes are configured such that the thermally triggeredexpression of recombinase causes them to be permanently switched on.This allows the biosynthetic cell to have a permanent response to asingle thermal trigger.

In some embodiments, in addition to placing the biosynthetic genes,transcription factors or recombinases downstream of thetemperature-dependent repressor's operator sequence, a circuit can bedesigned also having a copy of the temperature-sensitive repressoritself. This creates a negative feedback loop such that if there is anyleaky expression due to the low concentration of repressor protein, itwill lead to a buildup in the concentration of that protein. This isimplemented in our examples (e.g. FIG. 2A and FIG. 8F) where theexpression of the temperature-sensitive repressor is driven by both, itsown operator and a constitutively active (i.e. always on) promoter(pLacI).

In some embodiments, the temperature-responsive biosynthetic cellsherein described allow the biosynthetic activity of a cell to bemodified conveniently by changing the temperature of the bioreactor.This can be used, for example, to temporally control production, whereinone biosynthetic function operates for a short period to build up theappropriate chemical intermediate, then a second function is turned onto convert that intermediate into the next intermediate or product inthe synthetic pathway. Particular embodiments can be applied in caseswhere the two biosynthetic functions have cross-reactivity, preventingthem from effectively running at the same time.

In some embodiments, temperature-inactivated therapeutic cells aredescribed. Similar to the temperature-responsive therapeutic cellsherein described, in the temperature-inactivated therapeutic cells,instead of being directly regulated by the thermal transcriptionalrepressor, the therapeutic gene is controlled by another repressor (nottemperature dependent), which is, in turn controlled by the engineeredtemperature-dependent repressor. In this configuration, temperaturesabove the set-point of the thermal repressor will cause expression ofthe temperature-independent repressor, which will cause shut-down of theexpression of the therapeutic gene.

For example, as shown in FIGS. 8E-8G, TlpA controls the expression of atemperature-insensitive variant of the lambda repressor, which in turncontrols the expression of a fluorescent protein. This therapeutic cellis designed to turn off its therapeutic activity in response to athermal stimulus such as a fever, which could be an indicator ofundesired consequences for the host. Another example is applications ina topical cell therapy, which becomes active at the skin (set point 35°C.).

In some embodiments, instead of controlling the expression of atemperature-independent repressor, the thermal switches can be designedto control the expression of a recombinase, as described above. In thiscase, the therapeutic genes are flanked by recombinase sites in such amanner that the consequence of recombinase activity is to permanentlydisable their expression (e.g. by deleting them).

In some embodiments of the temperature-responsive therapeutic cells, thecells are designed to express therapeutic genes within a specifiedtemperature range (FIGS. 8E-8G). The thermal genetic circuit combinestwo orthogonal temperature-dependent transcriptional repressors, such asTlpA and TcI, as well as a temperature-independent version of therepressor that has a lower-temperature set point (e.g. cI in addition toTcI). The therapeutic gene is preceded by an operator that is repressedby both the lower-temperature set-point temperature-sensitive repressorand its temperature-independent analog. The temperature-independentrepressor is expressed under control of the higher-temperaturetranscriptional repressor.

As a result of this arrangement, there are three temperature dependenttherapeutic states. For example, if the lower-temperature repressor hasa set-point of X° C. and the higher-temperature repressor has aset-point of Y° C., such that X<Y, then the regimes are: i)temperature<X° C.: therapeutic genes are not expressed; ii)temperature>X ° C. and <Y ° C.; therapeutic genes are expressed; andiii) temperature>Y ° C.; therapeutic genes are not expressed. In thisconfiguration, the therapeutic cells become active when inside the host(e.g. X=36) and inactivated in response to a fever (e.g. Y=38.5).

Alternatively, the two temperature-dependent repressors can be used tocontrol the expression of two recombinases that switch the expression ofa given gene stably on and off.

In some embodiments of the temperature-responsive therapeutic cells, thetherapeutic cells are designed to express two different therapeuticgenes in response to two different temperatures. In this case, the cellcontains to different therapeutic genes under the independent control oftwo orthogonal temperature-dependent transcriptional repressors, eachwith a distinct thermal set-point (e.g. FIG. 8a-c ). In thisconfiguration, the therapeutic cells will be at the on-state when insidethe host (e.g. set-point of 36° C.) and become activated in response toa remote thermal signal such as focused ultrasound.

In some embodiments, a genetic circuit can be designed to be included ina temperature sensitive inactivable cell comprising a temperaturesensitive genetic circuit herein described in which at least onetemperature sensitive molecular component is configured to activate orinhibit at least one killer molecular component at an inactivatingbioswitch temperature Tbs₁ In some embodiments the inactivatingbioswitch temperature Tbs₁ is achieved in the temperature sensitivetherapeutic cell in response to a decrease in the cell temperatureassociated with a spatial translocation of the temperature sensitiveinactivable cell. In some embodiments at least one of the temperaturesensitive genetic molecular components comprises a coiled coil and/orglobular temperature sensitive transcription factor herein describedhaving a bioswitch temperature Tbs equal to Tbs_(L)

In some embodiments, the temperature sensitive killer circuit can bedesigned to include at least one temperature sensitive transcriptionalfactor activating at an inactivating temperature bioswitch Tbs₁ between32 to 45° C., in particular between 32° C. and 39° C., or between 32 to36° C. or 36 to 39° C., or 39 to 41° C., or above 41 C possibly between41 to 43° C., or 41 to 45° C.

In some those embodiments the output of the activated circuit can be theexpression of a protein that is toxic to the cell containing thecircuit, resulting in cell death, being unable to synthesize RNA orproteins or failing to divide. Example self-toxic output proteinsinclude CcdB and PezT. Alternatively, the output of the circuit could beshut-down of the expression of an antitoxin that is required to preventthe activity of an always-expressed toxin.

In particular in some of these embodiments, to enact cellular suicidewhen the temperature goes above a certain set-point, thetemperature-dependent repressor controls the expression of a suicidetoxin such as CcdB. To enact cellular suicide when the temperature goesbelow a certain set-point, the toxin from a toxin-antitoxin pair (e.g.CcdB) is expressed constitutively, and the anti-toxin (e.g. CcdA) isexpressed under the control of a temperature-dependent repressor. Whenthe temperature drops below the set-point, expression of the anti-toxinstops and the cell dies.

In some embodiments, the output of the killer circuit can be modulatedto in accordance with the characteristics of the target environment andother factors related to space and time of desired effects as well asfeatures of the cell as will be understood by a skilled person. Inparticular, the levels of toxin and antitoxin expression can be adjustedfor this circuit to function properly, for example, by fusing a proteindegradation tag to the antitoxin to accelerate cell death atnonpermissive temperatures.

In some of these embodiments, the temperature inactivable cells are alsotherapeutic cells and in particular, temperature sensitive therapeuticcells herein described that can be designed to destroy themselves in thecase of a fever (e.g. at set point 38.5° C.) or upon entry into the hostfrom being on the skin (set-point 35° C.). In some other embodiments,the therapeutic cells can be designed to destroy themselves afterleaving their host due to defecation, vomiting, cough, etc. Theset-point can be 36° C.

In particular, to prevent cells from surviving by mutating geneticcomponents of the circuit, the therapeutic cells can be designed tocontain multiple independent toxin (and, where appropriate, anti-toxin)systems regulated by one or more temperature-dependent transcriptionalrepressors. This redundancy exponentially decreases the likelihood ofescape.

In some embodiments one or more temperature sensitive transcriptionfactors, and in particular one or more coiled coil and/or globulartemperature sensitive transcription factors, vectors, genetic circuitand/or temperature sensitive cells herein described together with asuitable vehicle.

The term “vehicle” as used herein indicates any of various media actingusually as solvents, carriers, binders or diluents for the one or morecoiled coil and/or globular temperature sensitive transcription factors,vectors, genetic circuit and/or temperature sensitive cells hereindescribed that are comprised in the composition as an active ingredient.In particular, the composition including the one or more coiled coiland/or globular temperature sensitive transcription factors, vectors,genetic circuit and/or temperature sensitive cells herein described canbe used in one of the methods or systems herein described

In some embodiments, one or more temperature sensitive transcriptionfactors, and in particular one or more coiled coil and/or globulartemperature sensitive transcription factors, vectors, genetic circuitand/or temperature sensitive cells herein described and relatedcompositions can be used in a method to control a biological process inan individual or another target environment. The method comprisesadministering to the individual, or contacting the another targetenvironment with, one or more temperature sensitive cells hereindescribed comprising a temperature sensitive genetic circuit hereindescribed. In the method the temperature sensitive genetic circuit isconfigured to provide an output interfering with the biological processin the individual at a set target temperature between 34° C. and 44° C.

In some embodiments, the set target temperature can be selected from 34to 41<33° C. (ambient environment), 33-34° C. (including skintemperature) 34-36° C. (including hypothermic core temperature), 36-38°C. (including human physiological temperature), 38-40° C. (includingmild fever in humans, 40° C.-42° C. (including severe fever in humans),39° C.-45° C. (including applied hyperthermia in humans (e.g. HIFU))

In some embodiments, one or more temperature sensitive transcriptionfactors, and in particular one or more coiled coil and/or globulartemperature sensitive transcription factors, vectors, genetic circuitand/or temperature sensitive cells herein described and relatedcompositions can be used in a method to treat or prevent a condition inan individual. The method comprises administering to the individual oneor more therapeutic temperature sensitive cells herein describedcomprising a temperature sensitive genetic circuit herein described. Inthe method the temperature sensitive genetic circuit is configured toprovide a therapeutic output in the individual at a set targettemperature between 34° C. and 44° C.

The term “treatment” as used herein indicates any activity that is partof a medical care for, or deals with, a condition, medically orsurgically. The terms “treating” and “treatment” refer to reduction inseverity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage. Thus, forexample, “treating” a patient involves prevention of a symptom oradverse physiological event in a susceptible individual, as well asmodulation and/or amelioration of the status of a clinically symptomaticindividual by inhibiting or causing regression of a disorder or disease.

The term “prevention” as used herein with reference to a conditionindicates any activity which reduces the burden of mortality ormorbidity from the condition in an individual. This takes place atprimary, secondary and tertiary prevention levels, wherein: a) primaryprevention avoids the development of a disease; b) secondary preventionactivities are aimed at early disease treatment, thereby increasingopportunities for interventions to prevent progression of the diseaseand emergence of symptoms; and c) tertiary prevention reduces thenegative impact of an already established disease by restoring functionand reducing disease-related complications.

The term “condition” indicates a physical status of the body of anindividual (as a whole or as one or more of its parts e.g., bodysystems), that does not conform to a standard physical status associatedwith a state of complete physical, mental and social well-being for theindividual. Conditions herein described comprise disorders and diseaseswherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms in an individual.

The term “individual” or “subject” or “patient” as used herein in thecontext of treatment includes a single animal and in particular higheranimals and in particular vertebrates such as mammals and in particularhuman beings.

In some embodiments, a method of treating a disease is described. Themethod comprises administering the therapeutic cells described above toa subject, allowing for these cells to distribute within the body, andtreating the subject with a source of targeted thermal energy for atleast 1 minute that leads to activation of a thermal bioswitch. Thethermal energy can be in the form of focused ultrasound, radiofrequencymagnetic hyperthermia, microwave, infrared or contact heating (seeExample 4 and FIG. 9D). The methods herein described can be used totarget the therapeutic activity of the cells to a specific locationwithin the body, even if the cells are distributed more widely than thatone location.

In some embodiments, one or more temperature sensitive transcriptionfactors, and in particular one or more coiled coil and/or globulartemperature sensitive transcription factors, vectors, genetic circuitand/or temperature sensitive cells herein described and relatedcompositions can be used in a method to control cell viability in atemperature sensitive manner. The method comprises providing atemperature sensitive cell comprising one or more temperature sensitivegenetic circuits herein described comprising at least one temperaturesensitive molecular component configured to activate at least one killermolecular component at an inactivating bioswitch temperature Tbs₁, Themethod also comprises applying to the temperature sensitive cell theinactivating bioswitch temperature Tbs₁ for time and under condition toallow activation of the at least one killer components by the at leastone temperature sensitive molecular component and to result in death ofthe temperature sensitive cell.

In some embodiments, the deposition of thermal energy to activate thethermal bioswitch is guided spatially by magnetic resonance imaging(MRI). Additionally, MRI can be used to monitor the temperature of thetarget region and adjust the energy source to achieve the desired localtemperature (FIG. 9B).

Further details concerning the thermal bioswitches, and related thermalgenetic circuits, therapeutic cells, systems and methods of the presentdisclosure will become more apparent hereinafter from the followingdetailed disclosure of examples by way of illustration only withreference to an experimental section.

EXAMPLES

The tunable thermal bioswitches and related systems and methods hereindisclosed are further illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary methods andprotocols for preparing sets of polynucleotides and polypeptides,testing and characterizing these sets of polynucleotides andpolypeptides, building genetic circuits, and testing genetic circuits invivo and in vitro. A person skilled in the art will appreciate theapplicability and the necessary modifications to adapt the featuresdescribed in detail in the present section, to additional tunablethermal bioswitches, genetic circuits and therapeutic cells and relatedmethods and systems according to embodiments of the present disclosure.

Plasmid Construction and Molecular Biology. All constructs were made viarestriction cloning, KLD mutagenesis, or Gibson Assembly using enzymesfrom New England Biolabs. All plasmids and their sources of geneticmaterial are described in Table 1. All constructs were cloned in Mach1E. coli (Thermo Fisher) and the sequence-validated plasmids were assayedin NEB10β E. coli (NEB). Fluorescent reporters referred to in the textas GFP and RFP are mWasabi and mCherry, respectively [47, 48]. Allplasmids were constructed using the pETDuet-1 backbone (EMD Biosciences)with the relevant thermal biosensor elements replacing multiple cloningsites 1 and 2.

TABLE 1 Genetic constructs Plasmid Transcriptional Regulator(s) OutputGene Product(s) pTlpA-Wasabi TlpA mWasabi pTlpA- TlpA NonfluorescentmWasabi (S71T, Wasabi-NF G73A) pTcI-Wasabi TcI (cI852 Repressor, cIA67T) mWasabi pLacI241- LacI A241T (mutation made in pETDuet-1 mWasabiWasabi LacI) pLacI265- LacI G265D (mutation made in pETDuet- mWasabiWasabi 1 LacI) pTetR89- TetR A89D mWasabi Wasabi pTetR193- TetR I193NmWasabi Wasabi pLon-Wasasbi Lon Promoter (GenBank CP009072) mWasabipRpoH- RpoH Promoter (GenBank CP009072) mWasabi Wasabi pClp-WasabiClpP-ClpX Promoter (Genbank mWasabi CP009072) pHtpG-Wasabi HtpG Promoter(Genbank CP009072) mWasabi pDnaK- DnaK Promoter (Genbank CP009072)mWasabi Wasabi pGrpE-Wasabi GrpE Promoter (Genbank CP009072) mWasabipLacIq- LacIq Promoter mWasabi Wasabi pTlpA_(SP)- TlpA Promoter withputative Pribnow box mWasabi Wasabi scrambled pTlpA_(Reverse)- TlpAPromoter as reverse complement mWasabi Wasabi pTlpA₃₆- TlpA₃₆ mWasabiWasabi pTlpA₃₉- TlpA₃₉ mWasabi Wasabi pTcI₃₈-Wasabi TcI₃₈ mWasabipTcI₄₂-Wasabi TcI₄₂ mWasabi pCali2 TlpA₃₆, TcI mWasabi, mCherrypThermeleon TcI, TlpA, cI_(wt) (under control of TlpA) mWasabi, mCherrypKillswitch TlpA₃₆ CcdA with SsrA degradation tag Sources of geneticelements: TlpA: B. Finlay, Univ. British Columbia; mWasabi: F. Arnold,Caltech; mCherry: S. Qi, Stanford; CcdB: pLenti X1 Zeo DEST plasmid(Addgene #17299); TetR: pENTR1A plasmid (Addgene #22265); all otherelements: Gblock synthesis (IDT).

Table 2 shows a list of mutations in variants of TlpA and TcI.

TABLE 2 Mutations in variants of TlpA and TcI Nonsynonymous ConstructMutations Synonymous Mutations TlpA₃₆ P60L, D135V, K187R, K202I, L208QTlpA₃₉ D135V, A217V, L236F TcI₃₈ M1V, L65S, K68R, A50 (GCT −> GCC), E128F115L, D126G, D188G (GAG −> GAA), R129 (AGA −> AGG), T152 (ACA −> ACC),L185 (CTT −> CTC) TcI₄₂ K6N, S33T, Y61H, L51 (TTA −> CTA) L119P, F122C

Thermal Regulation Assay. 2 mL cultures of 2xYT media with 100 μg/mLampicillin were inoculated with a single colony per culture and grown at30° C., 250 rpm for 20 hours. After dilution to OD₆₀₀=0.1 in LB (Sigma)with 100 μg/mL ampicillin, the cells were propagated at 30° C., 250 rpmfor 1.5 hours, after which OD₆₀₀ was measured using a Nanodrop 2000c(Thermo Scientific) in cuvette mode every 10 minutes. At OD₆₀₀=0.25, thecultures were dispensed in 25 μL aliquots into 8 well PCR strips withoptically transparent caps (Bio-Rad) using a multichannel pipette andplaced into a spatial temperature gradient formed by a Bio-Rad C1000Touch thermocycler with the lid set to 50° C. The temperature in eachthermocycler well was verified using a TEF-30-T thermocouple (J-KEMScientific) immersed in 25 μL of pure water within a PCR tube. After theprescribed thermal stimulus, PCR strips were removed, vortexed, spundown on a tabletop centrifuge and the fluorescence was measured using aStratagene MX3005p qPCR (Agilent). Immediately after measurement, thecultures were diluted with 75 μL LB/Amp and mixed, after which 90 μL ofculture was transferred into 96 well plates (Costar black/clear bottom)for measurement of OD₆₀₀ using a SpectraMax M5 plate reader (MolecularDevices). For studies of gene expression as a function of thermalinduction time (FIGS. 2D-2F), samples were returned to incubation at 30°C. after their indicated thermal induction periods such that the totalexperimental duration was 24 hours. Fluorescence measurements were madeat the end of this period. Gene expression (E) was determined accordingto Equation 1:

$\begin{matrix}{E = {\frac{F_{sample} - F_{blank}}{{OD}_{sample} - {OD}_{blank}} - \frac{F_{background} - F_{blank}}{{OD}_{background} - {OD}_{blank}}}} & (1)\end{matrix}$

Here, F is the raw fluorescence of the given sample and OD is the OD ofthe given sample at 600 nm. Raw OD measurements for all experiments areprovided in FIGS. 1A-1H. As expected, bacterial growth is highest in thephysiological range of 35° C. to 39° C. The value of blank fluorescencewas determined as the average of all 96 wells in a qPCR plate filledwith 25 μL LB. Blank OD was taken as the y-intercept of a standard curveof 90 μL non-fluorescent E. coli cultures whose OD₆₀₀ values weredetermined by cuvette measurements in a Nanodrop 2000c spectrophotometer(96 samples total). Background fluorescence was measured from anon-fluorescent construct derived by mutating the chromophore of mWasabi[49] in the pTlpA-Wasabi plasmid (pTlpA-Wasabi-NF). Fluorescencemeasurements for the thermal expression landscapes of TlpA and TcI wereperformed using the plate reader due to signal saturation of the qPCR atthe 24 hour time point (Sample N=3; Background N=2 for each time pointand temperature). Errors from background measurements were propagated byaddition in quadrature. Errors from blank measurements were negligiblerelative to sample-to-sample variation (relative standard deviation<2%)and were omitted from the calculation.

Colony Screening for TlpA Tuning. Error-prone PCR was performed onpTlpA-Wasabi (Stratagene GeneMorph II kit) and on pTcI-Wasabi (NEB TaqPolymerase/0.2 mM MnCl₂) and the PCR products were inserted into theparent constructs using Gibson Assembly. The resulting libraries weretransformed into NEB10β E. coli and plated on LB Agar. Followingovernight incubation at 30° C. and the appearance of colonies, aReplica-Plating Tool (VWR 25395-380) was used to replicate each seedplate into two receiver plates. One receiver plate was grown overnightat the desired repressed temperature, and the other at the intendedactivation temperature. Upon the appearance of visible colonies, plateswere imaged in a Bio-Rad ChemiDoc MP imager using blue epifluorescentillumination and the 530/28 nm emission filter. Images were examinedmanually for colonies that appeared dark or invisible on the “off plate”but showed bright fluorescence on the “on plate”. Approximately 10³colonies were screened per library. These colonies were picked andsubjected to the liquid culture thermal activation assay describedabove, whereupon their thermal induction profile was compared to that oftheir parent plasmid. Variants that demonstrated sharp switching andlarge dynamic range between the desired new transition temperatures weresequenced, re-transformed, and assayed using a higher number ofreplicates.

In Vitro Toxin-Antitoxin Assays. NEB10β cells were transformed with thethermally regulated toxin-antitoxin plasmid and allowed to grow at 37°C. overnight. Because reversion of plasmids carrying toxic genes such asCcdB is known to be a common phenomenon, we used a replica plate screento isolate colonies that maintained a functional thermal kill switchafter transformation. To this end, we replica plated the originaltransformation into two new plates, one incubated at 25° C. and theother maintained at 37° C. Colonies that grew at the permissivetemperature of 37° C. and not at 25° C. were used in downstream in vitroor in vivo experiments. For in vitro experiments, the selected colonieswere grown in 2xYT media with 100 μg/mL ampicillin at 37° C. withshaking until OD₆₀₀ of 0.6 whereupon they were diluted and plated ontoLB agar plates. The plates were incubated overnight at either 25° C. or37° C., after which colony forming units (CFU) were counted.

Focused Ultrasound. MRI-guided focused ultrasound treatment wasperformed using a 16-channel ultrasound generator, motorizedMRI-compatible transducer positioning system and an annular arraytransducer operating at 1.5 MHz (Image Guided Therapy, Pessac, France).Targeting and real-time imaging was performed using a BrukerBiospec/Avance 7T MRI system with RF excitation delivered by a 7.2 cmdiameter volume coil and detection via a 3 cm diameter surface coil.Temperature monitoring was performed using a continuously applied FastLow Angle Shot sequence with a T_(R) of 75 ms and T_(E) of 2.5 ms,matrix size of 32×32, and varying FOVs as listed below. Phase imageswere processed in real time using ThermoGuide software (Image GuidedTherapy) and temperature was calculated from the per-pixel phaseaccumulation due to a decrease in proton precession frequency of 0.01ppm/° C. For in vitro heating, 100 μL of a saturated NEB10β cultureexpressing the temperature-inducible reporter circuit was platedovernight at 30° C. and incubated for approximately 12 hours to form alawn on a plate containing 0.24% w/v LB (Sigma) and 0.32% w/v Bacto Agar(BD). An approximately 3 cm×3 cm square of agar was excised from theplate and placed, with the bacterial side facing up, onto a comparablysized pad of 1 cm thick extra firm tofu (O Organics) coated with SCANultrasound gel (Parker Laboratories) to exclude air at the interface. A1 cm high plastic washer made by drilling through the lid of a VWR 35 mmplastic tissue culture dish was placed onto the bacteria and theassembly was inverted and placed onto the surface coil such that thebacterial lawn, facing down, was supported by the washer. The ultrasoundtransducer was positioned above the assembly, in contact with the tofuthrough another thin layer of ultrasound gel. To provide a reference tocompensate for global phase drift during the experiment, a second pieceof tofu was placed within the field of view but spatially separated by a1 cm air gap from the object under insonation. A fiber optic thermometer(Neoptix T1) was inserted into the reference tofu, and the differencebetween the MRI-derived reference temperature and thermometer-reportedtemperature was accounted for at the site of insonation when calculatingthe true focal heating. Ultrasound was applied with the focus aimed atthe tofu immediately adjacent to the agar layer with manual control ofpower level and duty cycle so as to maintain a temperature of 41.5-43°C. for 45 minutes. Imaging was performed as described above with amatrix size of 5.39×5.05 cm and a slice thickness of 2 mm. The plate wassubsequently returned to 30° C. for 5 hours and imaged using a Bio-RadChemiDoc MP imager with blue epi illumination and a 530/28 nm emissionfilter (mWasabi) and also green epi illumination and a 605/50 nm filter(mCherry).

Animal Procedures. All animal procedures were performed under a protocolapproved by the California Institute of Technology Institutional AnimalCare and Use Committee (IACUC). 9-week old BALB/c female mice and 4-weekold NU/J 2019 female mice were purchased from Jackson Laboratory (JAX);4-week old SCID/SHC female mice were purchased from Charles River. Forin vivo ultrasound actuation, E. coli expressing the pTlpA36-Wasabiplasmid were grown to OD 0.6, pelleted, and resuspended to OD 24. A 100μL bolus was injected subcutaneously into both hindlimbs of a nude mouse(SCID or NU/J2019). Mice were anaesthetized using a 2% isoflurane-airmixture and placed on a dedicated animal bed with the surface coilpositioned below the target limb of the mouse. Anesthesia was maintainedover the course of the ultrasound procedure using 1-1.5% isoflurane.Respiration rate was maintained at 20-30 breaths per minute andtemperature and respiration rate were continuously monitored using apressure pad (Biopac Systems) and a fiber optic rectal thermometer(Neoptix). The target limb was thermally activated by elevating thetemperature to 41° C. and maintaining the elevated temperature for 45min to 1 hour. Temperature monitoring and adjustment was performed asdescribed above for in vitro experiments. Following ultrasoundtreatment, the mouse was returned to its cage for four hours,anaesthetized, and imaged using a Bio-Rad ChemiDoc MP imager with blueepi illumination and the 530/28 nm emission filter (mWasabi). For hostfever sensing experiments, SCID mice injected with bacteria as describedabove were housed in an incubator preset to 41° C. for two hours andcontrol mice were housed at room temperature. Following treatment, allmice were housed at room temperature for four hours, anaesthetized, andimaged using a Bio-Rad ChemiDoc MP imager with blue epi illumination andthe 530/28 nm emission filter (mWasabi). Mouse images are representativeof three independent in vivo experiments. Fever-induced and control micewere littermates randomly selected for each experimental condition.Investigators were not blinded to group allocation because no subjectiveevaluations were performed. For host confinement experiments, BALB/cmice were given drinking water containing 0.5 mg/mL of ampicillin for 24h, and then starved for food overnight. E. coli were grown in 2xYT mediacontaining ampicillin at 37° C. with shaking until OD₆₀₀ of 0.6.Cultures were pelleted and resuspended at 10⁸ cells/mL in PBS containing1.5% NaHCO₃. 200 μL of the suspension was administered orally using agavage needle. Food was returned to the mice and the drinking watercontained ampicillin throughout the entire experiment. Fresh fecalsamples were collected from each mouse 5 hrs after gavage and incubatedat 37° C. or 25° C. for 24 h, then weighed, homogenized in PBS at 0.1g/mL, diluted and plated onto LB agar plates containing ampicillin.Plates were then incubated overnight at 25° C. and 37° C. Bacterialcolonies were counted as described above for in vitro toxin-antitoxinexperiments. The sample size was N=5 mice, which was chosen based onpreliminary experiments indicating that it would be sufficient to detectsignificant differences in mean values.

Electrophoretic Mobility Shift Assay. Interaction between TlpA, σ⁷⁰-RNApholoenzyme and DNA was demonstrated using a gel shift assay. For this,50 pmoles of fluorescein-labeled double stranded DNA representing theTlpA operator with flanking padding sequences (70 base pairs in total)was incubated with either 50 pmoles of TlpA protein or 5 Units (8.5pmoles) σ⁷⁰-RNAp holoenzyme (NEB M0551S) individually in 50 uL reactionbuffer comprising 40 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 0.01%Triton-X-100 and 1 mM DTT at a pH of 7.5. As a negative control, thewildtype TlpA operator was replaced with a scrambled version. Followingincubation at 37° C. for 30 minutes, 10 uL of the reaction mixture wassupplemented with glycerol to a final concentration of 5% and loaded ina nondenaturing 4% polyacrylamide resolving gel. The gel was run at 65 Vfor 90 minutes in buffer comprising 45 mM Tris-borate and 1 mM EDTA at apH of 8.3. DNA was visualized using Bio-Rad ChemiDoc MP imager usingblue epifluorescent illumination and the 530/28 nm emission filter.

Statistics and Replicates. Data is plotted and reported in the text asthe mean±SEM. Sample size is N=4 biological replicates in all in vitroexperiments unless otherwise stated. This sample size was chosen basedon preliminary experiments indicating that it would be sufficient todetect significant differences in mean values. P-values were calculatedusing a two-tailed unpaired heteroscedastic t-test.

Example 1: High-Performance Temperature Sensitive Transcription Factors

In order to engineer new families of robust, tunable, orthogonal thermalbioswitches, the performance of six temperature-dependenttranscriptional repressors and six endogenous heat shock promoters wascharacterized. The panel included TlpA, a transcriptional autorepressorfrom the virulence plasmid of Salmonella typhimurium. This proteincontains an approximately 300 residue C-terminal coiled-coil domain thatundergoes sharp, temperature-dependent uncoiling between 37° C. and 45°C., and an N-terminal DNA binding domain that, in its low-temperaturedimeric state, blocks transcription from the 52 bp TlpAoperator/promoter [1, 20]. In addition, a well-knowntemperature-sensitive variant of the bacteriophage λ repressor cI(mutant cI⁸⁵⁷, here referred to as TcI) acting on a tandem pR/pLoperator/promoter [50] was tested. In most previous applications, TcIrepression has been modulated via large changes in temperature (e.g.,steps from 30° C. to 42° C.) [50]. However, its original description asa virulence factor suggested that much sharper switching may be possible[51]. Alongside TlpA and TcI, four reported temperature-sensitivemutants of the E. coli repressors TetR (A89D and I193N) [52] and LacI(A241T and G265D) [53, 54] were tested, together with a panel ofendogenous heat shock promoters, including GrpE, HtpG, Lon, RpoH, Clpand DnaK (FIG. 2A).

The performance of these constructs is summarized in FIG. 2B. TlpA andTcI had by far the largest dynamic ranges (355±45 and >1,432,respectively), reflecting a combination of tighter repression atsub-threshold temperatures and stronger promoter activity abovethreshold. Both of these repressors show sharp thermal transitions, withgreater than 30-fold induction over ranges of 5° C. and 3° C. centeredat 43.5° C. and 39.5° C. for TlpA and TcI, respectively (FIG. 2C).Furthermore, both systems are capable of rapid induction, with greaterthan 10-fold changes in expression observed after a 1 hour thermalstimulus (FIG. 2D). Complete time-temperature induction profiles forTlpA and TcI are shown in FIGS. 2E-2F. In addition to their highperformance, TlpA and TcI are expected to be more orthogonal to cellularmachinery than both the native heat shock promoters and the engineeredTetR and LacI repressors, the latter of which are utilized in multipleendogenous and engineered gene circuits [22-24]. A homology search [25]showed that TlpA and TcI repressors are present in far fewer bacterialspecies than either TetR or LacI (FIGS. 3A-3B). Based on these factors,TlpA and TcI were chosen as starting points for further bioswitchengineering.

Since the TlpA operator/promoter has not been studied in E. coli, itsmolecular mechanisms were characterized to inform its utilization ingenetic circuits. As shown in FIGS. 4A-4E, the TlpA operator is a strongpromoter (88-fold stronger than LacI^(Q)) driven by the transcriptionfactor σ⁷⁰. Interestingly, this promoter has bidirectional activity withidentical thermal regulation in both orientations, but approximately200-fold lower maximal expression in the reverse direction (FIGS.4C-4D). This property will enable convenient adjustment ofTlpA-regulated expression according to circuit requirements.

Example 2: Tuning Bioswitch Activation Temperatures

Applications in microbial therapy require thermal bioswitches thatactivate at different transition temperatures. For example, a hostcolonization sensor should be activated at 37° C., while a feverdetector may work best with a thermal threshold of 39° C., and a focusedultrasound-activated switch may require a transition point of 41° C. toavoid nonspecific actuation. Synthetic biology applications outsidebiomedicine may likewise have a variety of thermal requirements. It isthus highly desirable to be able to tune thermal bioswitches to activateat new temperatures while retaining sharp, robust switching performance.To enable such tuning of TlpA and TcI, a simple and effectivehigh-throughput assay was devised based on colony fluorescence. E. coliexpressing GFP under the control of mutant repressors (generated byerror-prone PCR) was grown on solid media and the colonies werereplica-plated onto separate plates for simultaneous incubation atdesired “off” and “on” temperatures (FIG. 5A). Images of plates werethen taken with wide-field fluorescence, as shown in FIG. 6A. Asexpected, many colonies show constitutive expression (ostensibly due toloss of repressor function), while others fail to de-repress (mostlikely retaining their original high transition point). However, severalcolonies show thermal induction in the desired regime. Within eachscreen, several such colonies were selected to undergo liquid phasecharacterization of induction temperature, switching sharpness, andexpression levels (FIG. 6B). From these variants, mutants were selectedthat retained the desirable performance characteristics of the wild typerepressor, but with shifted transition temperatures.

Screening of TlpA mutants at off-on temperatures of 30-37° C. and 37-40°C. produced high-performance bioswitches centered at 36° C. and 39° C.,respectively, which were named TlpA₃₆ and TlpA₃₉ (FIG. 6C). For TcI,both downshifted (TcI₃₈, T_(m)=38° C.) and upshifted (TcI₄₂, T_(m)=42°C.) variants relative to the original protein were selected (FIG. 6D).Together, the engineered TlpA and TcI repressor families cover thebiomedically relevant range of 32° C. to 46° C. (FIG. 5B) whiledemonstrating a dynamic range similar to that of the wild type protein(Table 3). The amino acid substitutions identified in these bioswitchvariants are shown in FIGS. 7A-7J. The observed decrease in fluorescenceat the highest temperatures tested is possibly be due to thermalinstability of the cell's transcriptional and translational machinery.Remarkably, a single round of mutagenesis was sufficient in all cases toobtain at least one variant with the desired switching behavior,suggesting that both TlpA and TcI are highly tunable for a broad rangeof applications.

TABLE 3 Mutant and wild type bioswitch performance Variant Fold ChangeSEM (±) T_(off) T_(max) TlpA 355 45 31.4 44.6 TlpA₃₆ 370 63 31.4 44.6TlpA₃₉ 1523 434 31.4 44.6 TcI 1432 404 34.2 40 TcI₃₈ 1032 160 32.4 40TcI₄₂ 1692 444 32.4 45.7The reported T_(off) for each variant is the lowest temperature at whichfluorescence could be detected above noise. T_(max) is the temperatureat which fluorescence was maximal.

Example 3. Thermal Logic Circuits Using Orthogonal Bioswitches

To enable microbial therapy applications, it is useful to developthermal logic circuits capable of controlling multiple functions atdifferent temperatures or confining activity to within a narrow thermalrange. This would enable cells to, for example, initiate one therapeuticfunction upon host colonization and switch to a different functionduring a host fever response or local activation with focusedultrasound. It was hypothesized that since TlpA and TcI act onorthogonal target sequences, they could be combined in circuits designedfor multiplexed thermal control or band-pass activation of microbialfunction. To assess the first possibility (FIG. 8A), a construct wasmade encoding a GFP modulated by TlpA₃₆ and an RFP regulated by TcI(FIG. 8B). As predicted, upon exposure to a range of temperatures, thetwo reporter genes were activated independently at their expectedthresholds, with no apparent crosstalk in their induction (FIG. 8C).Independent thermal control of the co-expressed circuits is illustratedby spatially patterned bacterial variants incubated at 37° C. and 42° C.(FIG. 8D). Next, to develop a thermal band-pass filter (FIG. 8E), acircuit was engineered placing the expression of RFP under the controlof the lambda operator, gated by both TcI (turning on above 36° C.) andthe temperature-independent wild type cI repressor, which was itselfplaced under the control of TlpA (activating above 43° C.) as shown inFIG. 8F. The cI open reading frame was preceded by a T7 terminator and aweak ribosome binding site to reduce buildup of this repressor at 40-43°C. due to leakage of the upstream TlpA operon. This resulted in RFPexpression confined between 36 and 44° C., while simultaneously turningon GFP above RFP's turn-off temperature (FIGS. 8G-8H).

Example 4. Spatially Targeted Control Using Focused Ultrasound

After developing TlpA and TcI-based thermal bioswitches, their utilitywas demonstrated in three prototypical microbial therapy scenarios.First, the ability of thermal bioswitches to mediate spatially-selectivecontrol of microbial therapies using focused ultrasound was tested, amodality that is well established in its ability to elevate temperaturesin deep tissues with millimeter spatial precision [55] and utilizedclinically to treat diseases such as cancer [56] and essential tremor[57]. Focused ultrasound has been used to activate gene expression inmammalian cells [58], but apparently has not been employed to controlthe activity of microbes in vivo. Such control could be highlyadvantageous in applications where the activity of a systemicallyadministered microbial therapy needs to be localized to a specificanatomical site, such as a deep-seated tumor or section of thegastrointestinal tract, which would be difficult to reach withoptogenetic triggers. To test this concept, gene expression was firstactivated using focused ultrasound in tissue-mimicking phantoms underthe guidance of magnetic resonance imaging (MRI) [59] (FIG. 9A). Thisguidance enabled precise spatial targeting of the ultrasound focus andreal-time monitoring and adjustment of local temperature. This techniquewas first applied to a flat lawn of E. coli containing the multiplexedexpression circuit shown in FIG. 8B. This specimen was assembled with atissue-mimicking tofu phantom, and steady-state focal heating over 45minutes resulted in a radial thermal gradient with an average focaltemperature of 42° C., as observed by real-time MRI thermometry (FIG.9B). A corresponding pattern of spatially localized fluorescence is seenin FIG. 9C.

To establish the feasibility of this approach in vivo, E. coliexpressing GFP under the control of TlpA₃₆ were injected subcutaneouslyinto both hindlimbs of a nude mouse and MRI-guided focused ultrasoundwas applied to one location (FIG. 9D) to produce a local steady-statetemperature of 41° C. for 45 min to 1 hour. This thermal dose is belowthe damage thresholds for mammalian tissues such as muscle and brain[60, 61]. In vivo fluorescence imaging four hours after ultrasoundtreatment showed robust expression of GFP specifically at theultrasound-targeted anatomical site (FIG. 9E). TlpA₃₆ was selected asthe thermal bioswitch for these experiments because its activationthreshold is approximately 4° C. above the typical murine cutaneoustemperature [62], a sufficient difference for site-specific ultrasoundactivation.

Example 5. Programmed Responses to Mammalian Host Temperature

Thermal bioswitches can be used to develop autonomous thermosensitivemicrobes responsive to endogenous changes in host temperature. First, itwas investigated whether bacteria can be engineered to sense and respondto a host fever (FIG. 10A). One flank of a nude mouse was subcutaneouslyinjected with E. coli expressing GFP under the control of TlpA₃₆, andthe other flank with E. coli expressing GFP controlled by wild type TlpAas a high-threshold control for non-specific activation. The mouse wasthen housed at 41° C. for two hours in an established fever modelparadigm [63]. In vivo fluorescent imaging four hours after feverinduction shows robust expression of GFP in the flank injected withTlpA₃₆—regulated bacteria (FIG. 10B). No significant activation is seenin the opposite flank or in a mouse housed at room temperature (FIG.10C).

Second, it was tested whether a thermal bioswitch operating at 37° C.could be used to confine the activity of genetically engineered microbesto the in vivo environment of a mammalian host and thereby limit thepotential for environmental contamination. Towards this end, a geneticcircuit was designed in which TlpA₃₆ controls the expression of CcdA, abacterial antitoxin, while constitutively expressing the toxin CcdB,thereby restricting growth to temperatures above 37° C. (FIG. 10D). Adegradation tag was fused to CcdA to accelerate cell death atnon-permissive temperatures. Bacteria carrying this plasmid grewnormally above this permissive temperature, while bacteria incubated at25° C. had significantly reduced survival as demonstrated by their CFUcounts in FIG. 10E. These bacteria were administered to mice by oralgavage and fecal pellets were collected after five hours to allowtransit through the gastrointestinal tract. The pellets were kept for 24hours at either 25° C., corresponding to excretion into the ambientenvironment, or at 37° C., equivalent to persistent residence in thegut, and subsequently assayed for colony formation. The survival ofcells excreted into ambient temperature was reduced by ten thousand foldcompared to cells maintained under host conditions (FIG. 10F).

Example 6. Calculation of Hill Coefficient

Circular dichroism (CD) spectra were recorded with a Aviv 62DS.Acquisition parameters were set as follows: spectral width 300-200 nm,scanning speed 50 nm/min, bandwidth 2 nm, in RNA-free conditions, in 1×Phosphate buffered saline, pH 7.4. CD melting curves were recorded witha temperature slope of 1° C./min at a wavelength of 222 nm between 25°C. and 50° C. The CD melting curves were normalized and fitted accordingto Hill's equation (Eq. 2).

FIGS. 23A-C show the CD melting curves normalized and fitted accordingto Hill's equations for TlpA-CC WT (FIG. 23A), TlpA-CC-DED (FIG. 23B),and DED-TlpA-CC (FIG. 23C). “DED” is a charged amino acid sequence(Asp-Glu-Asp) appended to the N-terminus of the TlpA coiled-coil domainin DED-TlpA-CC and appended to the C-terminus of the TlpA coiled-coildomain in TlpA-CC-DED. The CD melting curve was also normalized andfitted for tropomysin for comparison (FIG. 23D). The proteins wereprovided at a concentration of 10 The calculated Hill coefficients arelisted in Table 4.

TABLE 4 Hill coefficients of TlpA, TlpA variants and Tropomyosin HillName Coefficient TlpA_CC_wt_10uM 19.1475 TlpA_CC_DED_10uM 16.7159DED_TlpA_CC_10uM 24.7294 Tropomyosin 9.7109

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the materials, compositions, systems andmethods of the disclosure, and are not intended to limit the scope ofwhat the inventors regard as their disclosure. Those skilled in the artwill recognize how to adapt the features of the exemplified methods andarrangements to additional genetic circuit, related nodes, molecularcomponents, sets of polynucleotides, polypeptides and/or metabolites, inaccording to various embodiments and scope of the claims.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence. Further, the computer readable form of the sequence listingof the ASCII text file P1984-USC-Seq-List-ST25 is incorporated herein byreference in its entirety.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thedisclosure has been specifically disclosed by embodiments, exemplaryembodiments and optional features, modification and variation of theconcepts herein disclosed can be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. The term “plurality” includestwo or more referents unless the content clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified may beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein may be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably maybe practiced in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the invention and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe devices, device components, methods steps set forth in the presentdescription. As will be obvious to one of skill in the art, methods anddevices useful for the present methods may include a large number ofoptional composition and processing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

REFERENCES

-   1. Hurme, R., et al., DNA binding exerted by a bacterial gene    regulator with an extensive coiled-coil domain. J Biol Chem, 1996.    271(21): p. 12626-31.-   2. Marr, M. T. and J. W. Roberts, Promoter recognition as measured    by binding of polymerase to nontemplate strand oligonucleotide.    Science, 1997. 276(5316): p. 1258-60.-   3. Koski, P., et al., A new alpha-helical coiled coil protein    encoded by the Salmonella typhimurium virulence plasmid. J Biol    Chem, 1992. 267(17): p. 12258-65.-   4. Lupas, A., M. Van Dyke, and J. Stock, Predicting coiled coils    from protein sequences. Science, 1991. 252(5009): p. 1162-4.-   5. McDonnell, A. V., et al., Paircoil 2: improved prediction of    coiled coils from sequence. Bioinformatics, 2006. 22(3): p. 356-8.-   6. Vincent, T. L., P. J. Green, and D. N. Woolfson,    LOGICOIL—multi-state prediction of coiled-coil oligomeric state.    Bioinformatics, 2013. 29(1): p. 69-76.-   7. Grigoryan, G. and A. E. Keating, Structural specificity in    coiled-coil interactions. Curr Opin Struct Biol, 2008. 18(4): p.    477-83.-   8. Stayrook, S., et al., Crystal structure of the lambda repressor    and a model for pairwise cooperative operator binding. Nature, 2008.    452(7190): p. 1022-5.-   9. Kyte, J. and R. F. Doolittle, A simple method for displaying the    hydropathic character of a protein. Journal of molecular    biology, 1982. 157(1): p. 105-132.-   10. Lam, K. S., Mini-review. Application of combinatorial library    methods in cancer research and drug discovery. Anti-cancer drug    design, 1997. 12(3): p. 145-167.-   11. Chappell, J., et al., The centrality of RNA for engineering gene    expression. Biotechnology Journal, 2013. 8(12): p. 1379-1395.-   12. Berman, H. M., et al., The protein data bank. Acta    Crystallographica Section D: Biological Crystallography, 2002.    58(6): p. 899-907.-   13. Lohse, M. B., et al., Identification and characterization of a    previously undescribed family of sequence-specific DNA-binding    domains. Proceedings of the National Academy of Sciences, 2013.    110(19): p. 7660-7665.-   14. Flynn, R. L. and L. Zou, Oligonucleotide/oligosaccharide-binding    fold proteins: a growing family of genome guardians. Critical    Reviews in Biochemistry and Molecular Biology, 2010. 45(4): p.    266-275.-   15. Chen, X., J. L. Zaro, and W.-C. Shen, Fusion protein linkers:    property, design and functionality. Advanced drug delivery    reviews, 2013. 65(10): p. 1357-1369.-   16. Ye, Y. and A. Godzik, Flexible structure alignment by chaining    aligned fragment pairs allowing twists. Bioinformatics, 2003.    19(suppl 2): p. ii246-ii255.-   17. Maiti, R., et al., SuperPose: a simple server for sophisticated    structural superposition. Nucleic acids research, 2004. 32(suppl    2): p. W590-W594.-   18. Gelly, J.-C., et al., iPBA: a tool for protein structure    comparison using sequence alignment strategies. Nucleic acids    research, 2011. 39(suppl 2): p. W18-W23.-   19. Ilinkin, I., J. Ye, and R. Janardan, Multiple structure    alignment and consensus identification for proteins. BMC    bioinformatics, 2010. 11(1): p. 1.-   20. Hurme, R., et al., A proteinaceous gene regulatory thermometer    in Salmonella. Cell, 1997. 90(1): p. 55-64.-   21. Piraner, D. I., et al., Tunable thermal bioswitches for in vivo    control of microbial therapeutics. Nature Chemical Biology, 2016.-   22. Wilson, C. J., et al., The lactose repressor system: paradigms    for regulation, allosteric behavior and protein folding. Cell Mol    Life Sci, 2007. 64(1): p. 3-16.-   23. Bertram, R. and W. Hillen, The application of Tet repressor in    prokaryotic gene regulation and expression. Microb Biotechnol, 2008.    1(1): p. 2-16.-   24. Jensen, P. R., H. V. Westerhoff, and O. Michelsen, The use of    lac-type promoters in control analysis. Eur J Biochem, 1993.    211(1-2): p. 181-91.-   25. Altschul, S. F., et al., Gapped BLAST and PSI-BLAST: a new    generation of protein database search programs. Nucleic acids    research, 1997. 25(17): p. 3389-3402.-   26. Drozdetskiy, A., et al., J Pred4: a protein secondary structure    prediction server. Nucleic acids research, 2015: p. gkv332.-   27. Lauck, F., et al., RosettaBackrub—a web server for flexible    backbone protein structure modeling and design. Nucleic acids    research, 2010. 38(suppl 2): p. W569-W575.-   28. Kuhlman, B., et al., Design of a novel globular protein fold    with atomic-level accuracy. science, 2003. 302(5649): p. 1364-1368.-   29. Humphris, E. L. and T. Kortemme, Prediction of protein-protein    interface sequence diversity using flexible backbone computational    protein design. Structure, 2008. 16(12): p. 1777-1788.-   30. Renfrew, P. D., et al., Incorporation of noncanonical amino    acids into Rosetta and use in computational protein peptide    interface design. PLoS One, 2012. 7(3): p. e32637.-   31. Pabo, C. O. and M. Lewis, The operator-binding domain of lambda    repressor: structure and DNA recognition. Nature, 1982. 298: p.    443-447.-   32. Hu, J. C., et al., Sequence requirements for coiled-coils:    analysis with repressor-GCN4 leucine zipper fusions. Science, 1990.    250(1400): p. 0.-   33. Mangan, S. and U. Alon, Structure and function of the feed    forward loop network motif. Proc Natl Acad Sci USA, 2003.    100(21): p. 11980-5.-   34. Elowitz, M. B. and S. Leibler, A synthetic oscillatory network    of transcriptional regulators. Nature, 2000. 403(6767): p. 335-8.-   35. Hooshangi, S., S. Thiberge, and R. Weiss, Ultrasensitivity and    noise propagation in a synthetic transcriptional cascade. Proc Natl    Acad Sci USA, 2005. 102(10): p. 3581-6.-   36. Gardner, T. S., C. R. Cantor, and J. J. Collins, Construction of    a genetic toggle switch in Escherichia coli. Nature, 2000.    403(6767): p. 339-42.-   37. Shin, J. and V. Noireaux, An E. coli cell-free expression    toolbox: application to synthetic gene circuits and artificial    cells. ACS Synth Biol, 2012. 1(1): p. 29-41.-   38. Ganguly, T., et al., A point mutation at the C-terminal half of    the repressor of temperate mycobacteriophage L1 affects its binding    to the operator DNA. BMB Reports, 2004. 37(6): p. 709-714.-   39. Hemrich, J., et al., The cl repressor of bacteriophage P1    operator-repressor interaction of wild-type and mutant repressor    proteins. Nucleic acids research, 1989. 17(19): p. 7681-7692.-   40. Vogel, J., et al., Temperature-sensitive mutations in the    bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding    domain. Journal of bacteriology, 1991. 173(20): p. 6568-6577.-   41. Servant, P., C. Grandvalet, and P. Mazodier, The RheA repressor    is the thermosensor of the HSP18 heat shock response in Streptomyces    albus. Proceedings of the National Academy of Sciences, 2000.    97(7): p. 3538-3543.-   42. Kamp, H. D. and D. E. Higgins, A protein thermometer controls    temperature-dependent transcription of flagellar motility genes in    Listeria monocytogenes. PLoS Pathog, 2011. 7(8): p. e1002153.-   43. Chao, Y. P., et al., Construction and characterization of    thermo-inducible vectors derived from heat-sensitive lacI genes in    combination with the T7 A1 promoter. Biotechnology and    bioengineering, 2002. 79(1): p. 1-8.-   44. Wissmann, A., et al., Selection for Tn10 tet repressor binding    to tet operator in Escherichia coli: isolation of    temperature-sensitive mutants and combinatorial mutagenesis in the    DNA binding motif. Genetics, 1991. 128(2): p. 225-232.-   45. Herbst, K., et al., Intrinsic thermal sensing controls    proteolysis of Yersinia virulence regulator RovA. PLoS Pathog, 2009.    5(5): p. e1000435.-   46. Huang, D., W. J. Holtz, and M. M. Maharbiz, A genetic bistable    switch utilizing nonlinear protein degradation. Journal of    biological engineering, 2012. 6(1): p. 1.-   47. Ai, H. W., et al., Hue-shifted monomeric variants of Clavularia    cyan fluorescent protein: identification of the molecular    determinants of color and applications in fluorescence imaging. BMC    Biol, 2008. 6: p. 13.-   48. Shaner, N.C., et al., Improved monomeric red, orange and yellow    fluorescent proteins derived from Discosoma sp. red fluorescent    protein. Nat Biotechnol, 2004. 22(12): p. 1567-72.-   49. Wielgus-Kutrowska, B., et al., Folding and unfolding of a non    fluorescent mutant of green fluorescent protein. Journal of Physics:    Condensed Matter, 2007. 19(28): p. 285223.-   50. Valdez-Cruz, N. A., et al., Production of recombinant proteins    in E. coli by the heat inducible expression system based on the    phage lambda pL and/or pR promoters. Microb Cell Fact, 2010. 9: p.    18.-   51. Sussman, R. and F. Jacob, Sur un systeme de repression    thermosensible chez le bacteriophage lambda d'Escherichia coli.    Comptes rendus hebdomadaires des séances de l'Académie des sciences,    1962(254): p. 1517-1519.-   52. Wissmann, A., et al., Selection for Tn10 tet repressor binding    to tet operator in Escherichia coli: isolation of    temperature-sensitive mutants and combinatorial mutagenesis in the    DNA binding motif. Genetics, 1991. 128(2): p. 225-32.-   53. Chao, Y. P., et al., Construction and characterization of    thermo-inducible vectors derived from heat-sensitive lacI genes in    combination with the T7 A1 promoter. Biotechnol Bioeng, 2002.    79(1): p. 1-8.-   54. McCabe, K. M., et al., LacI(Ts)-regulated expression as an in    situ intracellular biomolecular thermometer. Appl Environ    Microbiol, 2011. 77(9): p. 2863-8.-   55. Haar, G. T. and C. Coussios, High intensity focused ultrasound:    physical principles and devices. Int J Hyperthermia, 2007. 23(2): p.    89-104.-   56. Al-Bataineh, O., J. Jenne, and P. Huber, Clinical and future    applications of high intensity focused ultrasound in cancer. Cancer    treatment reviews, 2012. 38(5): p. 346-353.-   57. Elias, W. J., et al., A pilot study of focused ultrasound    thalamotomy for essential tremor. New England Journal of    Medicine, 2013. 369(7): p. 640-648.-   58. Deckers, R., et al., Image-guided, noninvasive, spatiotemporal    control of gene expression. Proceedings of the National Academy of    Sciences, 2009. 106(4): p. 1175-1180.-   59. Fite, B. Z., et al., Magnetic resonance thermometry at 7T for    real-time monitoring and correction of ultrasound induced mild    hyperthermia. PloS one, 2012. 7(4): p. e35509.-   60. McDannold, N. J., et al., Usefulness of MR Imaging-Derived    Thermometry and Dosimetry in Determining the Threshold for Tissue    Damage Induced by Thermal Surgery in Rabbits 1. Radiology, 2000.    216(2): p. 517-523.-   61. McDannold, N., et al., MRI investigation of the threshold for    thermally induced blood-brain barrier disruption and brain tissue    damage in the rabbit brain. Magnetic resonance in medicine, 2004.    51(5): p. 913-923.-   62. Rudaya, A. Y., et al., Thermoregulatory responses to    lipopolysaccharide in the mouse: dependence on the dose and ambient    temperature. Am J Physiol Regul Integr Comp Physiol, 2005.    289(5): p. R1244-52.-   63. Pritchard, M. T., et al., Protocols for simulating the thermal    component of fever: preclinical and clinical experience.    Methods, 2004. 32(1): p. 54-62.

1.-20. (canceled)
 21. A method to engineer a TlpA dimer having TlpAtemperature sensing domain Tm₀ defining a bioswitch temperature Tbs₀with Tbs₀=Tm₀+0° C. to 5° C., the TlpA dimer comprising two TlpAmonomer, each having SEQ ID NO: 488 and comprising a TlpA DNA bindingdomain of SEQ ID NO: 472 and a TlpA coiled coil temperature sensingdomain having a temperature sensing amino acid sequence comprisingalpha-helical heptad repeats having SEQ ID NOs: 473 to 484 and SEQ iDNO; 486, each alpha-helical heptad repeat further having a register a,b, c, d, e, f, or g, in which up to 5 consecutive amino acid residuesare optionally missing, the method comprising replacing in the TlpAcoiled coil temperature sensing domain of at least one TlpA monomer, atleast one of a polar amino acid in at least one of position b, positione, and position g of at least one alpha-helical heptad repeat, with ahydrophobic amino acid; an amino acid in at least one of position a andposition d of at least one alpha-helical heptad repeat, with an aminoacid configured to increase or decrease hydrophobic packing betweenamino acid residues in positions a and/or d of correspondingalpha-helical heptad repeats in the TlpA dimer, and an amino acid in atleast one of position e and g of at least one alpha-helical heptadrepeat with an amino acid configured to increase or decrease coulombicrepulsion between corresponding amino acid residues in positions a, d, eand/or g of corresponding heptad repeats in the TlpA dimer variant; thereplacing performed to obtain an engineered TlpA dimer variant having amelting temperature Tm_(m) lower or higher than Tm₀ and a bioswitchtemperature Tbs_(m) lower or higher than Tbs₀.
 22. The method of claim21, wherein the replacing is performed to replace a polar amino acid ina position b of at least one alpha-helical heptad repeat of thealpha-helical heptad repeats having SEQ ID NOs: 473 to 484 and SEQ iDNO; 486, with a hydrophobic amino acid and wherein the replacingperformed to obtain an engineered TlpA dimer variant having a Tbs_(m)lower than Tbs₀.
 23. The method of claim 22, wherein the hydrophobicamino acid is selected from alanine, valine, leucine, isoleucine,proline, phenylalanine, tryptophan, cysteine and methionine.
 24. Themethod of claim 21, wherein the replacing is performed to replace ahydrophobic amino acid in a position d of at least one alpha-helicalheptad repeat of the alpha-helical heptad repeats having SEQ ID NOs: 473to 484 and SEQ iD NO; 486, with a polar amino acid and wherein thereplacing performed to obtain an engineered TlpA dimer variant having aTbs_(m) lower than Tbs₀.
 25. The method of claim 24, wherein the polaramino acid is selected from serine (Ser), threonine (Thr), asparagine(Asn), glutamine (Gln), and tyrosine (Tyr).
 26. The method of claim 21,wherein the replacing is performed to replace a first charged amino acidin a position e of at least one alpha-helical heptad repeat of thealpha-helical heptad repeats having SEQ ID NOs: 473 to 484 and SEQ IDNO; 486, with a second charged amino acid having a pKa which differsfrom the pKa of the first charged amino acid by equal or higher than0.5. and wherein the replacing is performed to obtain an engineered TlpAdimer variant having a Tbs_(m) lower than Tbs₀.
 27. The method of claim24, wherein the second charged amino acid is selected from lysine,arginine and histidine.
 28. The method of claim 21, wherein thereplacing is performed to replace at least one of an hydrophobic aminoacid in a position a, an hydrophobic amino acid in a position d, inplace of a hydrophobic amino acid residue, a charged amino acid in aposition e, and a charged amino acid in a position g, of at least onealpha-helical heptad repeat of the alpha-helical heptad repeats havingSEQ ID NOs: 473 to 484 and SEQ ID NO; 486, with an amino acid configuredso that pairs formed by corresponding residues in positions a, d, e, andg of a resulting alpha-helical heptad repeats of a resulting geneticallyengineered TlpA monomer variants interact with a coulombic force F≥1 pN,wherein $\begin{matrix}{F = {k_{e}\frac{q_{1}q_{2}}{r^{2}}}} & {{Eq}.\mspace{14mu}(4)}\end{matrix}$ where k_(e) is Coulomb's constant (k_(e)=8.99×10⁹ N m²C⁻²), q₁ and q₂ are the signed magnitudes of the charges on each aminoacid residue of the pair of residues, and the scalar r is the distancebetween the charges and wherein the replacing is performed to obtain anengineered TlpA dimer variant having a Tbs_(m) lower than Tbs₀.
 29. Themethod of claim 28, wherein the charged amino acid is lysine and theamino acid configured so that pairs interact with a coulombic force F≥1pN is arginine.
 30. The method of claim 21, wherein the replacing isperformed to the replace at least one of a hydrophobic amino acid in aposition a, with a polar amino acid, a hydrophobic amino acid in aposition d, with a polar amino acid, of at least one alpha-helicalheptad repeat of the alpha-helical heptad repeats having SEQ ID NOs: 473to 484 and SEQ ID NO; 486, and wherein the replacing is performed toobtain an engineered TlpA dimer variant having a Tbs_(m) higher thanTbs₀.
 31. The method of claim 30, serine (Ser), threonine (Thr),asparagine (Asn), glutamine (Gln), and tyrosine (Tyr).
 32. The method ofclaim 21 wherein the replacing is performed to replace at least one of ahydrophobic amino acid in a position a, a hydrophobic amino acid in aposition d, a charged amino acid in a position e, and a charged aminoacid in a position g of at least one alpha-helical heptad repeat of thealpha-helical heptad repeats having SEQ ID NOs: 473 to 484 and SEQ IDNO; 486, with an amino acid configured so that pairs formed bycorresponding residues in positions a, d, e, and g on the alpha-helicalheptad repeats of the genetically engineered TlpA monomer variantsinteract with a coulombic force F≥1 pN calculated according to$\begin{matrix}{F = {k_{e}\frac{q_{1}q_{2}}{r^{2}}}} & {{Eq}.\mspace{14mu}(4)}\end{matrix}$ where k_(e) is Coulomb's constant (k_(e)=8.99×10⁹ N m²C⁻²), q₁ and q₂ are the signed magnitudes of the charges on each aminoacid residue of the pair of residues, and the scalar r is the distancebetween the charges and wherein the replacing is performed to obtain anengineered TlpA dimer variant having a Tbs_(m) higher than Tbs₀.
 33. Themethod of claim 21, wherein the hydrophobic amino acid is selected fromalanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan, cysteine and methionine.
 34. The method of claim 21, furthercomprising engineering at least one TlpA monomer to include one or moreinsertions, deletions and/or replacements within a percent variationfrom 0% to 20% along the total length of the sequences.
 35. The methodof claim 21, wherein the bioswitch temperature Tbs_(m) is Tm_(m)+2.5 to5° C.
 36. The method of claim 21, wherein the bioswitch temperatureTbs_(m) is Tm_(m)+1 to 3.5° C.
 37. The method of claim 21, wherein thebioswitch temperature Tbs_(m) is Tm_(m)+0 to 1.5° C.
 38. The method ofclaim 21, wherein the Tbs_(m) is 32° C. to 46° C.
 39. The method ofclaim 21, wherein the Tbs_(m) is from 36° C. to 38° C. or from 38° C. to40° C.
 40. The method of claim 21, wherein the Tbs_(m) is from 38° C. to40° C.
 41. The method of claim 21, wherein the engineered TlpA dimervariant has two engineered TlpA monomers each having sequence SEQ ID NO:463.
 42. The method of claim 21, wherein the engineered TlpA dimervariant has two engineered TlpA monomers each having sequence SEQ ID NO465.